Methods and compositions for DNA profiling

ABSTRACT

Embodiments disclosed herein provide methods for constructing a DNA profile comprising: providing a nucleic acid sample, amplifying the nucleic acid sample with a plurality of primers that specifically hybridize to at least one target sequence comprising a SNP and at least one target sequence comprising a tandem repeat, and determining the genotypes of the at least one SNP and at least one tandem repeat in the amplification products, thereby constructing the DNA profile of the nucleic acid sample. Embodiments disclosed herein further provide a plurality of primers that specifically hybridize to at least one short target sequence and at least one long target sequence in a nucleic acid sample, wherein amplifying the nucleic acid sample using the plurality of primers in a single reaction results in a short amplification product and a long amplification product, wherein each of the plurality of primers comprises one or more tag sequences.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 14/622,632, filed Feb. 13, 2015 (now U.S. Pat. No. 10,422,002, issued Sep. 24, 2019), which application claims priority to U.S. Provisional Application No. 62/103,524 filed Jan. 14, 2015, U.S. Provisional Application No. 62/043,060 filed Aug. 28, 2014, and U.S. Provisional Application No. 61/940,942 filed Feb. 18, 2014, the contents of which are incorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled IP-1192-US_SL.txt, created Sep. 16, 2019, which is identical to the sequence listing filed on Mar. 7, 2018, in application Ser. No. 14/622,632, which is 59,789 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

Embodiments provided herein relate to methods and compositions for DNA profiling. Some embodiments relate to methods of amplification of target sequences of variant sizes in a single reaction, followed by subsequent sequencing of the library.

BACKGROUND OF THE DISCLOSURE

Historically, the use of a subset of markers in a human genome has been utilized to determine an individual's personal identity, or DNA fingerprint or profile. These markers include locations or loci of short tandem repeated sequences (STRs) and intermediate tandem repeated sequences (ITRs) which in combination are useful in identifying one individual from another on a genetic level. The analysis of these markers has become standardized in the analysis of DNA found at crime scenes. For example, in the United States a number of these repeated sequences have been combined to create a Combined DNA Index System (CODIS), which serve as the laboratory standard for DNA profiling in criminal cases. Other countries similarly have adopted a standard system for DNA profiling. These systems have also been utilized to determine paternity and familial relationships. However, the current systems are all based on size separation of these repeated loci on an electrophoretic system and are thus limited to the number of loci that can be differentiated in such a system. For example, some of the current commercial systems for DNA profiling for forensics purposes differentiate only 16 markers due to the limitations of the electrophoretic detection methods.

SUMMARY OF THE DISCLOSURE

Embodiments relate to systems and methods that are not content limited and that bring different pieces of genetic information about an individual together to provide a comprehensive, more complete DNA profile of an individual. The present disclosure describes methods and compositions that enable this profile of an individual, thereby advancing the fields of personal and forensic genomics.

DNA profiling currently uses selected biological markers for determining the identity of a DNA sample. For example, the most common analysis for determining a DNA profile is to determine the profile for a number of short tandem repeated (STRs) sequences found in an organism's genome. The analysis consists of amplifying defined STR sequences that can be up to 400 bp long that can be differentiated by size on an electrophoretic gel or by using capillary electrophoresis (CE). Electrophoresis is used to detect size changes due to differences in the number of repeated STRs at a given locus and as such the length of the PCR amplicons, which for the CE system is between 50-500 bp. To help overcome the limits imposed by size differentiation methodologies (i.e., STRs of overlapping amplicon size cannot be differentiated), current methods of DNA profiling utilize different sets of labelled primers such that amplicons that overlap in size can be labelled with different fluorescent dyes whereon, upon excitation, the emission spectra differ thereby allowing for overlapping amplicons to be differentiated using differences in the dye excitation and emission spectra. Using differentiated labeling, current methods allow for the multiplexing of 24 different STR loci using 6 differently detectable dyes in one DNA profiling run.

There are many limitations to the current DNA profiling methodologies. As previously mentioned, size differentiated systems limit the number of loci that can be discretely determined at a given time. Another limitation of the established methods for DNA profiling is that the DNA to be analyzed oftentimes is degraded and the size range of some of the markers does not accommodate degraded DNA, for example the amplicons can be larger than the size of the fragments of the degraded DNA For degraded DNA, amplicons of 400 bp are considered very long and can result in loss of amplification of those longer loci. When DNA analysts amplify degraded DNA samples to identify their STR profile, for example a sample found at a crime scene, oftentimes they are unable to detect all the loci resulting in a partial profile which can make matching a suspect in a crime scene to a crime sample difficult or impossible. As a default with such samples, a DNA analyst has little choice and if any sample is left over, additional assays need to be performed to identify other markers which might give a clue as to the identity of the individual, such as single nucleotide polymorphisms (SNPs), mini-STRs, or mitochondrial DNA (mtDNA) analysis. However, precious sample must be expended on each assay with no certainty of success in finally identifying an individual. FIG. IA demonstrates the potential different paths to DNA identification, all of which are separate workflows and require aliquots of precious samples. When one or more simple workflows need to be combined and potentially repeated multiple times then the resulting process is no longer simple or an effective use of a precious sample.

Embodiments described in the present application provide methods, compositions and systems for determining the DNA profile of an individual or organism by next generation sequencing (NGS) thereby providing a solution to the problems and limitations of current methodologies for DNA profiling. FIG. 1B shows an exemplary workflow of the disclosed methods in one embodiment. Disclosed herein are methods and compositions for combining a multitude of forensically relevant markers into one assay including, but not limited to, short tandem repeats (STRs), intermediate tandem repeats (ITRs), identity informative single nucleotide polymorphisms (iSNPs), ancestry informative single nucleotide polymorphisms (aSNPs) and phenotypic informative single nucleotide polymorphisms (pSNPs).

The present disclosure describes assays that overcome the limitations of current methodologies for DNA profiling. Disclosed embodiments provide methods and compositions for multiplex amplification, library preparation and sequencing of combined STRs, ITRs, iSNPs, aSNPs, and pSNPs from one nucleic acid sample in a single multiplex reaction. Disclosed methods analyze a plurality of markers in one experimental assay with minimal sample handling, using low amounts of sample DNA including degraded DNA some embodiments described can be utilized for databanking DNA profiles and/or DNA profiles that can be used for criminal casework. Some embodiments provide PCR methods and compositions developed to be sensitive enough to detect sub-nanogram amounts of DNA Further, the unconventional primer design parameters allow for highly multiplexed PCR for the identification of STRs, ITRs and SNPs in one multiplex reaction. For criminal casework, the present methods and compositions incorporate unique molecule identifiers (UMIs) which aide in removal of, for example, PCR and sequencing errors, stutter and the like from sequencing results. See Kivioj a et al., Nat. Meth. 9, 72-74 (2012). As well, the results from the methods and compositions disclosed herein are compatible with existing databases.

Therefore, embodiments disclosed herein provide methods for constructing a DNA profile comprising: providing a nucleic acid sample, amplifying the nucleic acid sample with a plurality of primers that specifically hybridize to at least one target sequence comprising a single nucleotide polymorphism (SNP) and at least one target sequence comprising a tandem repeat in a multiplex reaction to generate amplification products, and determining the genotypes of the at least one SNP and the at least one tandem repeat in the amplification products, thereby constructing the DNA profile of the nucleic acid sample.

In some embodiments, the methods comprise generating a nucleic acid library from the amplification products. In some embodiments, the methods comprise determining the sequences of the nucleic acid library. In some embodiments, the nucleic acid sample is from a human. In some embodiments, the nucleic acid sample is from an environmental sample, a plant, a non-human animal, a bacterium, archaea, a fungus, or a virus. In some embodiments, the DNA profile is used for one or more of disease diagnostics or prognosis, cancer biomarker identification, genetic anomaly identification or genetic diversity analysis. In some embodiments, the DNA profile is used for one or more of databanking, forensics, criminal case work, paternity or personal identification. In some embodiments, the at least one SNP indicates the ancestry or a phenotypic characteristic of the source of the nucleic acid sample. In some embodiments, each of the plurality of primers has a low melting temperature and/or has a length of at least 24 nucleotides. In some embodiments, each of the plurality of primers has a melting temperature that is less than 60 degrees C. In some embodiments, each of the plurality of primers has a melting temperature that is about 50 degrees C. to about 60 degrees C. In some embodiments, each of the plurality of primers has a length of at least 24 nucleotides. In some embodiments, each of the plurality of primers has a length of about 24 nucleotides to about 38 nucleotides. In some embodiments, each of the plurality of primers comprises a homopolymer nucleotide sequence. In some embodiments, the nucleic acid sample is amplified by polymerase chain reaction (PCR). In some embodiments, the nucleic acid sample is amplified in an amplification buffer having a salt concentration that is increased compared to the salt concentration of an amplification buffer used in conjunction with conventionally designed primers. In some embodiments, the salt comprises KCl, LiCl, NaCl, or a combination thereof. In some embodiments, the salt comprises KCl. In some embodiments, the concentration of KCl in the amplification buffer is about 100 mM to about 200 mM. In some embodiments, the concentration of KCl in the amplification buffer is less than about 150 mM. In some embodiments, the concentration of KCl in the amplification buffer is about 145 mM. In some embodiments, the SNP is an ancestry SNP, a phenotypic SNP, an identity SNP, or a combination thereof. In some embodiments, the plurality of primers specifically hybridize to at least 30 SNPs. In some embodiments, the plurality of primers specifically hybridize to at least 50 SNPs. In some embodiments, the tandem repeat is a short tandem repeats (STR), an intermediate tandem repeat (ITR), or a variant thereof. In some embodiments, the plurality of primers specifically hybridize to at least 24 tandem repeat sequences. In some embodiments, the plurality of primers specifically hybridize to at least 60 tandem repeat sequences. In some embodiments, the nucleic acid sample comprises about 100 pg to about 100 ng DNA

In some embodiments, the nucleic acid sample comprises about 10 pg to about 100 pg DNA In some embodiments, the nucleic acid sample comprises about 5 pg to about 10 pg DNA In some embodiments, the nucleic acid sample comprises genomic DNA In some embodiments, the genomic DNA is from a forensic sample. In some embodiments, the genomic DNA comprises degraded DNA In some embodiments, at least 50% of the genotypes of the at least one SNP and at least one tandem repeat are determined. In some embodiments, at least 80% of the genotypes of the at least one SNP and at least one tandem repeat are determined. In some embodiments, at least 90% of the genotypes of the at least one SNP and at least one tandem repeat are determined. In some embodiments, at least 95% of the genotypes of the at least one SNP and at least one tandem repeat are determined. In some embodiments, each of the plurality of primers comprises one or more tag sequences. In some embodiments, the one or more tag sequences comprise a primer tag, a capture tag, a sequencing tag, a unique molecular identifier tag, or a combination thereof. In some embodiments, the one or more tag sequences comprise a primer tag. In some embodiments, the one or more tag sequences comprise a unique molecular identifier tag.

Embodiments disclosed herein provide methods of constructing a nucleic acid library comprising: providing a nucleic acid sample, and amplifying the nucleic acid sample with a plurality of primers that specifically hybridize to at least one target sequence comprising a single nucleotide polymorphism (SNP) and at least one target sequence comprising a tandem repeat sequence in a multiplex reaction to generate amplification products.

In some embodiments, the nucleic acid sample is not fragmented prior to the amplification. In some embodiments, the target sequences are not enriched prior to the amplification. In some embodiments, the at least one SNP indicates the ancestry or a phenotypic characteristic of the source of the nucleic acid sample. In some embodiments, each of the plurality of primers comprises one or more tag sequences. In some embodiments, the one or more tag sequences comprise a primer tag, a capture tag, a sequencing tag, or a unique molecular identifier tag, or a combination thereof. In some embodiments, the methods include amplifying the amplification products with a second plurality of primers. In some embodiments, each of the second plurality of primers comprises a portion corresponding to the primer tag of the plurality of primers and one or more tag sequences. In some embodiments, the one or more tag sequences of the second plurality of primers comprise a capture tag, or a sequencing tag, or a combination thereof. In some embodiments, the methods include adding single stranded-binding protein (SSB) to the amplification products. In some embodiments, the nucleic acid sample and/or the amplification products are amplified by polymerase chain reaction (PCR). In some embodiments, the nucleic acid sample and/or the amplification products are amplified in an amplification buffer having a salt concentration that is increased compared to the salt concentration of an amplification buffer used in conjunction with conventionally designed primers. In some embodiments, the salt comprises KCl, LiCl, NaCl, or a combination thereof. In some embodiments, the salt comprises KCl. In some embodiments, the concentration of KCl in the amplification buffer is about 100 mM to about 200 mM. In some embodiments, the concentration of KCl in the amplification buffer is less than about 150 mM. In some embodiments, the concentration of KCl in the amplification buffer is about 145 mM.

Embodiments disclosed herein provide a nucleic acid library comprising a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprise at least one tandem repeat sequence flanked by a first pair of tag sequences and at least one single nucleotide polymorphism (SNP) sequence flanked by a second pair of tag sequences. Further provided is a nucleic acid library constructed using the methods and compositions disclosed herein. In some embodiments, the at least one SNP indicates the ancestry or a phenotypic characteristic of the source of the plurality of nucleic acid molecules.

Embodiments disclosed herein provide a plurality of primers that specifically hybridize to at least one short target sequence and at least one long target sequence in a nucleic acid sample, wherein amplifying the nucleic acid sample using the plurality of primers in a single multiplex reaction results in at least one short amplification product and at least one long amplification product, wherein each of the plurality of primers comprises one or more tag sequences.

In some embodiments, the short target sequence comprises a single nucleotide polymorphism (SNP) and the long target sequence comprises a tandem repeat. In some embodiments, the one or more tag sequences comprise a primer tag, a capture tag, a sequencing tag, a unique molecular identifier tag, or a combination thereof. In some embodiments, each of the plurality of primers has a low melting temperature and/or has a length of at least 24 nucleotides. In some embodiments, each of the plurality of primers has a melting temperature that is less than 60 degrees C. In some embodiments, each of the plurality of primers has a melting temperature that is about 50 degrees C. to about 60 degrees C. In some embodiments, each of the plurality of primers has a length of at least 24 nucleotides. In some embodiments, each of the plurality of primers has a length of about 24 nucleotides to about 38 nucleotides. In some embodiments, each of the plurality of primers comprises a homopolymer nucleotide sequence. In some embodiments, the nucleic acid sample is amplified by polymerase chain reaction (PCR). In some embodiments, the SNP is an ancestry SNP, a phenotypic SNP, an identity SNP, or a combination thereof In some embodiments, the plurality of primers specifically hybridize to at least 30 SNPs. In some embodiments, the plurality of primers specifically hybridize to at least 50 SNPs. In some embodiments, the tandem repeat is a short tandem repeats (STR), an intermediate tandem repeat (ITR), or a variant thereof. In some embodiments, the plurality of primers specifically hybridize to at least 24 tandem repeat sequences. In some embodiments, the plurality of primers specifically hybridize to at least 60 tandem repeat sequences.

Embodiments disclosed herein provide kits comprising at least one container means, wherein the at least one container means comprises a plurality of primers disclosed herein.

In some embodiments, the kits include a reagent for an amplification reaction. In some embodiments, the reagent is an amplification buffer for polymerase chain reaction (PCR). In some embodiments, the amplification buffer comprises a salt concentration that is increased compared to the salt concentration of an amplification buffer used in conjunction with conventionally designed primers. In some embodiments, the salt comprises KCl, LiCl, NaCl, or a combination thereof. In some embodiments, the salt comprises KCl. In some embodiments, the concentration of KCl in the amplification buffer is about 100 mM to about 200 mM. In some embodiments, the concentration of KCl in the amplification buffer is less than about 150 mM. In some embodiments, the concentration of KCl in the amplification buffer is about 145 mM.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A and FIG. 1B show the differences in the A) current workflow for DNA profiling versus B) that of one exemplary embodiment of the present disclosure.

FIG. 2 shows one exemplary embodiment of a method for creating a library useful for DNA profiling.

FIG. 3 shows another exemplary embodiment of a method for creating a library useful for DNA profiling.

FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D are line graphs illustrating electropherogram results of how a primer pair designed by traditional methods and following established PCR primer design protocols and limitations can cause non-specific amplification of genomic targets and obscuration of desired amplicons detection when combined with primers designed following methods of the present disclosure; A) 10 primer pairs designed by methods of the present disclosure directed to SNP loci, B) and D) the 10 primers plus an additional primer pair designed by traditional methods demonstrating that the additional primer pair interferes with the 10 primer pairs during amplification, and C) the 10 primer pairs plus an additional primer pair, wherein the additional primer pair was also designed by following the methods of the present disclosure resulting in a successful amplification of all the targeted SNPs. The X-axis is the size of the library fragments (bp) and the Y axis is fluorescence units of (FU) of the amplified peaks of the amplified fragments.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D and FIG. 5E are box plots showing exemplary results for an experiment following the workflow outlined in FIG. 2 , which was used to identify a panel of 56 STRs and mix of 75 identity-informative SNPs (iSNPs), ancestry-informative SNPs (aSNPs) and phenotypic-informative SNPs (pSNPs) in a multiplex amplification and sequencing reaction from a sample. Reported are replicated results demonstrating successful amplification and sequencing of the STR loci from the panel; A) box plot demonstrating intra-locus balance for 25 heterozygous STRs from the panel, B) box plot demonstrating low stutter for the majority of the 56 STR loci, C) box plot demonstrating sequencing coverage for the STR loci, D) box plot demonstrating the sequence coverage for the SNPs, and E) box plot demonstrating balance for 22 heterozygous SNPs from the panel. The lower error bar indicates the minimum value, the upper error bar indicates the maximum value, the lower box reports the 25th percentile and the upper box reports the 75th percentile with the mean being the intersection between the lower and upper boxes.

FIG. 6 shows a series of bar charts showing the exemplary STR loci plots from the experiment of FIG. 5 . The plots show different allelic calls for the STRs in the panel of FIG. 5 .

FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D and FIG. 7E are box plots showing exemplary results for an experiment following the workflow outlined in FIG. 3 , which was used to identify a panel of 26 STRs and a mix of 94 iSNPs, aSNPs and pSNPs in a multiplex amplification and sequencing reaction from a sample. Reported are replicated results demonstrating successful amplification and sequencing of the STRs from the panel; A) box plot demonstrating intra-locus balance for 21 heterozygous STR loci from the panel, B) box plot demonstrating low stutter for the 26 STR loci (39 of the 47 alleles of the 26 loci showed no stutter), C) box plot demonstrating sequencing coverage for the STR loci (read numbers normalized using the UMIs), D) box plot demonstrating the sequence coverage for the SNPs and E) box plot demonstrating balance for 21 heterozygous iSNPs from the panel.

The lower error bar indicates the minimum value, the upper error bar indicates the maximum value, the lower box reports the 25th percentile and the upper box reports the 75th percentile with the mean being the intersection between the lower and upper boxes.

FIG. 8 shows a series of bar charts showing the exemplary STR loci plots from the experiment of FIG. 7 . The plots show different allelic calls for the STRs in the panel of FIG. 7 .

FIG. 9 shows bar graphs of samples analyzed without UMIs, and with UMIs. The left panel for each set represents samples analyzed without UMIs and the right panel for each set represents samples analyzed with UMIs. The X axis designates the repeat number of the STR and the Y axis designates the count number of the particular allele. The error lines within the bars separate the sequencing error (upper part of the bar) from the correct sequence (lower part of the bar) within the STR sequence.

FIG. 10A and FIG. 10B show exemplary results from an experiment where the DNA ratio was 90:10 female: male. A) a subset of STR loci call results for STR loci when using current capillary electrophoresis DNA profiling methods, and B) several STR loci call results for several STR loci when using the methods of the present application. Both the CE methods and the methods of the present application did detect the low level of male DNA contamination.

FIG. 11 shows bar charts that show that STR loci specific to the Y chromosome were detected in the experiment of FIG. 9 , further demonstrating that the present application can detect contaminating male DNA and specific STR loci from that male DNA whereas two experiments would need to be run with the current CE methodologies to do so.

FIG. 12 is a table that shows exemplary high level sequencing results from an experiment using 12 sample individuals and a reference individual, demonstrating consistency of STR and SNP calls between two replications.

FIG. 13 is a table that shows exemplary population statistics from the experiment shown in FIG. 12 .

FIG. 14 is a table that shows exemplary phenotype predictions based on genotype of pSNPs from the experiment shown in FIG. 12 .

FIG. 15 is a graph showing exemplary ancestry mapping based on genotype of aSNPs from the experiment shown in FIG. 12 .

FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D and FIG. 16E are bar charts showing the exemplary STR loci plots from the experiment of FIG. 12 .

FIG. 17A and FIG. 17B are bar charts showing exemplary SNP plots from the experiment of FIG. 12 .

FIG. 18A and FIG. 18B show box plots showing the intra-locus balance of exemplary STR and SNP loci from the experiment of FIG. 12 .

FIG. 19A and FIG. 19B are graphs showing stutter analysis of exemplary STR loci from the experiment of FIG. 12 .

FIG. 20 is a table that shows exemplary isometric heterozygotes in STR loci from the experiment of FIG. 12 . Sequences represented include Sample 3/Locus D9S1122/Repeat Number 13 (TAGATAGATAGATAGATAGATAGATAGATAGA TAGATAGATAGATAGATAGA) (SEQ ID NO: 405); Sample 3/Locus D9S1122/Repeat Number 13 (TAGATCGATAGATAGATAGATAGATAGATAGATAGATAGATAGAT AGATAGA) (SEQ ID NO: 406); Sample 7/D2S1338/Repeat Number 19 (TGCCTGCCT GCCTGCCTGCCTGCCTGCCTGCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTC CT TCCTTCCTTCC) (SEQ ID NO: 407); Sample 7/D2S1338/Repeat Number 19 (TGCCTGCCTGCCTGCCTGCCTGCCTGCCTTCCTTCCTTCCTTCCTTCCTTC CTTCCTTCCTTCCTTCCTTCCTTCC) (SEQ ID NO: 408); Sample 13/D8S1179/Repeat Number 14 (TCTATCTATCTGTCTATCTATCTATCTATCTATCTATCTATCTATCTA TCTATCTA) (SEQ ID NO: 409); Sample 13/D8S1179/Repeat Number 14 (TCTATCTGTCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTA) (SEQ ID NO: 410); Sample 14/D8S1179/Repeat Number 13 (TCTATCTATCTGTCTA TCTATCTATCTATCTATCTATCTATCTATCTATCTA (SEQ ID NO: 411); Sample 14/D8S1179/Repeat Number 13 (TCTATCTGTCTATCTATCTATCTATCTATCTATCTA TCTATCTATCTATCTA (SEQ ID NO: 412); Sample 15/D2S1338/Repeat Number 19 (TGCCTGCCTGCCTGCCTGCCTGCCTGCCTTCCTTCCTTCCTTCCTTCCTTCCTT CC TTCCTTCCTTCCTTCCTTCC) (SEQ ID NO: 413); Sample 15/D2S1338/Repeat Number 19 (TGCCTGCCTGCCTGCCTGCCTGCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCT TCCTTCCTTCCTTCCTTCCTTCC) (SEQ ID NO: 414); Sample 17/D8S1179/Repeat Number 13 (TCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTAT CTA) (SEQ ID NO: 415); Sample 17/D8S1179/Repeat Number 13 (TCTATCTG TCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTA) (SEQ ID NO: 416); Sample 17/D9S1122/Repeat Number 12 (TAGATAGATAGATAGATAGATAGAT AGATAGATAGATAGATAGATAGA) (SEQ ID NO: 417); Sample 17/D9S1122/Repeat Number 12 (TAGATCGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGA) (SEQ ID NO: 418); 2800M/D9S1122/Repeat Number 12 (TAGATAGATAGATAGATAGA TAGATAGATAGATAGATAGATAGATAGA) (SEQ ID NO: 419); 2800M/D9S1122/Repeat Number 12 (TAGATCGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGA) (SEQ ID NO: 420).

FIG. 21 is a block diagram that shows an exemplary inheritance plot based on variants within the STR D8S1179 from the experiment of FIG. 12 . Sequences represented include [TCTA]2 [TCTG]1 [TCTA]9 (SEQ ID NO: 421); [TCTA]2 [TCTG]1 [TCTA]10 (SEQ ID NO: 422); [TCTA]13 (SEQ ID NO: 423); [TCTA]1 [TCTG]1 [TCTA]11 (SEQ ID NO: 424); [TCTA]10 (SEQ ID NO: 425).

FIG. 22 is a block diagram that shows an exemplary inheritance plot based on variants within the STR D13S317 from the experiment of FIG. 12 . Sequences represented include [TATC]1 1 [AATC]2 (SEQ ID NO: 426); [TATC]12 [AATC]2 (SEQ ID NO: 427); [TATC]1 1 TATCAATC (SEQ ID NO: 428); [TATC]12 TATCAATC(SEQ ID NO: 429).

FIG. 23 is a table that shows exemplary genotyping results using degraded DNA.

FIG. 24A and FIG. 24B show exemplary STR genotyping results and intro-locus balance at difference DNA inputs.

FIG. 25A and FIG. 25B show exemplary SNP genotyping results and intro-locus balance at difference DNA inputs.

DETAILED DESCRIPTION Definitions

All patents, applications, published applications and other publications referred to herein are incorporated by reference to the referenced material and in their entireties. If a term or phrase is used herein in a way that is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the use herein prevails over the definition that is incorporated herein by reference.

As used herein, the singular forms “a”, “an”, and “the” include plural references unless indicated otherwise, expressly or by context. For example, “a” dimer includes one or more dimers, unless indicated otherwise, expressly or by context.

As used herein, the terms “DNA profile,” “genetic fingerprint,” and “genotypic profile” are used interchangeably herein to refer to the allelic variations in a collection of polymorphic loci, such as a tandem repeat, a single nucleotide polymorphism (SNP), etc. A DNA profile is useful in forensics for identifying an individual based on a nucleic acid sample. DNA profile as used herein may also be used for other applications, such as diagnosis and prognosis of diseases including cancer, cancer biomarker identification, inheritance analysis, genetic diversity analysis, genetic anomaly identification, databanking, forensics, criminal case work, paternity, personal identification, etc.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule” are used interchangeably herein to refer to a polymeric form of nucleotides of any length, and may comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”).

As used herein, “sequence identity” or “identity” or “homology” in the context of two nucleotide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. The portion of the nucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

As used herein, “substantially complementary or substantially matched” means that two nucleic acid sequences have at least 90% sequence identity. Preferably, the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. Alternatively, “substantially complementary or substantially matched” means that two nucleic acid sequences can hybridize under high stringency condition(s).

It is understood that aspects and embodiments of the invention described herein include “consisting” and/or “consisting essentially of” aspects and embodiments.

Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.

Methods for Constructing a DNA Profile

Established methodologies for determining the DNA profile are limited in a number of ways. For example, current methods detect size changes of amplified loci which differ due to changes in lengths of tandem repeated sequences found in a DNA sample. To multiplex STR amplifications for visualization, the amplifications have to be designed to space the different amplicons sizes within the size separation limits of the electrophoretic system, which for CE is from about 50-500 bp. As such, only a limited number of the repeated sequences can be visualized in one assay. For example, the GLOBALFILER™ PCR amplification kit (APPLIED BIOSYSTEMS) PCR amplification kit) is reportedly able to differentiate 24 STR loci by using 6 different dyes. Further, such methods have issues when sample DNA is degraded as is common with DNA samples from a crime scene, such that longer amplification products are not possible resulting in an incomplete DNA profile. Current methods are also oftentimes not sensitive enough to detect small amounts of contaminating DNA so a mixed sample can go undetected and unreported, which could be critical for criminal casework. As such, current methods can lead to incomplete results which lead to inconclusive results, which can be detrimental for DNA profiling.

Additionally, current targets do not include information about sample ancestry, phenotypic traits such as possible eye color and other individualized sample information. Some sequencing methodologies have attempted to include both STR and SNP detection. For example, library preparation followed by custom enrichment for STRs and SNPs has been attempted, however not all STRs are completely covered as library preparation methods typically involve sample shearing that can obliterate the targeted sequence. Further, established primer design methods and protocols can provide primer sets for amplifying long sequences (e.g., STRs) or short sequences (e.g., SNPs), but the combinations of both in one reaction have not met with success.

The present disclosure describes solutions to the problems and limitations of the current DNA profiling systems. Methods and compositions described herein allow for the combination of STRs and SNPs into one assay using PCR to amplify the targets and generate libraries for sequencing. While developing the present assays, it was unexpectedly discovered that, for example, when utilizing unconventional and counterintuitive primer design, both STRs and SNPs can be amplified in one reaction which allows the sequence for all targeted loci to be determined. Surprisingly, when designing amplification primers using parameters contrary to the current dogma surrounding primer design, primers were created that allowed for the longer STR regions to be amplified and the short SNP regions to be amplified in a more or less balanced manner thereby allowing for both STRs and SNPs to be multiplex amplified together.

The methods and compositions disclosed herein for determining the DNA profile of an organism can be used whenever differently sized sets of amplicons are desired from one amplification reaction outside of DNA profiling. For example, if targets of interest for PCR include both large gene regions and short SNP regions which may result in amplicons that vary in size from hundreds to thousands of base pairs versus amplicons of less than 100 base pairs, respectively, then the methods and compositions described herein could allow for successful simultaneous amplification of the gene and SNP targets which would not have been possible without practicing the disclosed methods. Further, the methods and compositions disclosed herein may apply to any organism, for example humans, non-human primates, animals, plants, viruses, bacteria, fungi and the like. As such, the present methods and compositions are not only useful for DNA profiling (e.g., forensics, paternity, individual identification, etc.) and humans as a target genome, but could also be used for other targets such as cancer and disease markers, genetic anomaly markers and/or when the target genome is not human based.

Therefore, embodiments disclosed herein provide methods for constructing a DNA profile comprising: providing a nucleic acid sample, amplifying the nucleic acid sample with a plurality of primers that specifically hybridize to at least one target sequence comprising a single nucleotide polymorphism (SNP) and at least one target sequence comprising a tandem repeat, and determining the genotypes of the at least one SNP and at least one tandem repeat in the amplification products, thereby constructing the DNA profile of the nucleic acid sample.

It would be appreciated by those skilled in the art that any suitable techniques may be used in determining the genotypes of the target sequences including, but not limited to, array-based hybridization, sequencing, or the like. Therefore, in some embodiments, the methods disclosed herein may comprise generating a nucleic acid library, such as a sequencing library, from the amplification products, and determining the sequences of the nucleic acid library.

In some embodiments, the present disclosure provides methods and compositions for DNA profiling that comprise the concurrent identification of STRs and iSNPs, for example for use in population or personal databanking. In such databanks, personal data is not necessarily needed as the individuals are typically known. However, if additional information is desired then additional information targets can be added for concurrent identification. Short tandem repeats are well known in the art, and consist of repeated di- or tri nucleotide sequences. Intermediate tandem repeats are typically considered repeated sequences of between 4 to 7 nucleotide sequences. SNPs utilized herein can be of any form that might offer insight into a person's physical characteristics. Those exemplified herein are SNPs that provide clues for ancestry or heritage (aSNPs) and those that provide clues for phenotypic characteristics (phenotypic-informative SNPs). In methods described herein, a DNA profile assay might include any number of these SNPs in combination with STR and ITR loci determinations.

For example, the present disclosure provides additional methods and compositions where, along with STRs and iSNPS, additional targets are included. If more information about an individual is desired, for example when a sample belongs to an unknown individual or group of individuals as can be the case for criminal casework, the other information markers can be added to the STR and iSNPs, such as SNPs related to ancestry (aSNPs) and SNPs related to phenotypic variants (phenotypic-informative SNPs). The additional information can then be used to aid investigators, for example, by providing insight into an unknown individual's heritage, eye color, hair color, and the like. As such, the addition of all the combined information can provide a more complete DNA profile of an individual that was not previously known using current methods of DNA profiling.

The methods and compositions disclosed herein are designed to be sensitive enough to detect sub-nanogram amounts of nucleic acid molecules. Further, the methods and compositions disclosed herein may be useful to amplify a nucleic acid sample made having low-quality nucleic acid molecules, such as degraded and/or fragmented genomic DNA from a forensic sample. The nucleic acid sample may be a purified sample or a crude DNA containing lysate, for example derived from a buccal swap, paper, fabric or other substrate that may be impregnated with saliva, blood, or other bodily fluids. As such, in some embodiments, the nucleic acid sample may comprise low amounts of, or fragmented portions of DNA, such as genomic DNA For example, the nucleic acid sample may comprise an amount of nucleic acid (e.g., genomic DNA) that is, is about, or is less than, 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 11 pg, 12 pg, 13 pg, 14 pg, 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 10 ng, 100 ng, or is in a range defined by any two of these values, for example, 10 pg to 100 pg, 10 pg to 1 ng, 100 pg to 1 ng, 1 ng to 10 ng, 10 ng to 100 ng, etc. In some embodiments, the nucleic acid sample may comprise an amount of nucleic acid (e.g., genomic DNA) that is about 100 pg to about 1 ng. In some embodiments, the nucleic acid sample may comprise an amount of nucleic acid (e.g., genomic DNA) that is more than about 62.5 pg. In some embodiments, additional fragmentation steps, such as sonication or endonuclease digestion, are not included in the fragmentation procedures.

In some embodiments, the methods and compositions disclosed herein are capable of successfully determining the genotypes of one or more of the target sequences, for example, SNPs, STRs, etc., even with sub-nanogram amounts of and/or degraded nucleic acid samples. For example, the methods and compositions disclosed herein are capable of successfully determining the genotype that is, is about, or is more than, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between any two of the above values, of the target sequences. In some embodiments, the methods and compositions disclosed herein are capable of successfully determining the genotype of more than about 50%, 80%, 90%, 95%, 98% or more of the target sequences. In some embodiments, the methods and compositions disclosed herein are capable of achieve an intra-locus balance of more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between any two of the above values, of the target sequences.

For forensic investigation, the plurality of primers may incorporate unique molecule identifiers (UMIs) which aide in removal of, for example, PCR and sequencing errors, stutter and the like from sequencing results. See Kivioja et al., supra. As discussed in further detail elsewhere in this disclosure, inclusion of UMI in the primers also allows the identification of variants within tandem repeat loci, further enhancing the usefulness of the current methods and compositions for DNA profiling and other purposes such as inherence analysis.

Accordingly, in some embodiments, the genotypes of the tandem repeat sequences as disclosed herein may include sequence variants within the tandem repeat loci. Therefore, a homozygote for a tandem repeat (e.g., 13, 13 for D9S1122) using the traditional method may be identified as an isometric heterozygote based on sequence variants within the tandem repeat. As would be appreciated by those skilled in the art, taking into account the intra-locus sequence variants would greatly enhance the usefulness of the methods disclosed herein, for example, for inheritance analysis.

Methods for Constructing a Nucleic Acid Library

Embodiments disclosed herein provide methods of constructing a nucleic acid library comprising: providing a nucleic acid sample, and amplifying the nucleic acid sample with a plurality of primers that specifically hybridize to at least one target sequence comprising a single nucleotide polymorphism (SNP) and at least one target sequence comprising a tandem repeat sequence.

The methods and compositions disclosed herein are designed to be sensitive enough to detect sub-nanogram amounts of nucleic acid molecules. Further, the methods and compositions disclosed herein may be useful to amplify a nucleic acid sample that consists of low-quality nucleic acid molecules, such as degraded and/or fragmented genomic DNA from a forensic sample. The nucleic acid sample may be either purified or a crude DNA containing lysate, for example derived from a buccal swap, paper, fabric or other substrate that may be impregnated with saliva, blood, or other bodily fluids. As such, in some embodiments, the nucleic acid sample may comprise low amount of or fragmented DNA, such as genomic DNA For example, the nucleic acid sample may comprise an amount of nucleic acid (e.g., genomic DNA) that is, is about, or is less than, 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 11 pg, 12 pg, 13 pg, 14 pg, 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 10 ng, 100 ng, or is in a range defined by any two of these values, for example, 10 pg to 100 pg, 10 pg to 1 ng, 100 pg to 1 ng, 1 ng to 10 ng, 10 ng to 100 ng, etc. In some embodiments, the nucleic acid sample may comprise an amount of nucleic acid (e.g., genomic DNA) that is about 100 pg to about 1 ng. In some embodiments, the nucleic acid sample may comprise an amount of nucleic acid (e.g., genomic DNA) that is more than about 62.5 pg. In some embodiments, additional fragmentation steps, such as sonication or endonuclease digestion, are not included.

In some embodiments, methods disclosed herein comprise amplification and library preparation in anticipation of downstream parallel sequencing. An assay may include two PCR master mixes, two thermostable polymerases, two primer mixes and library adaptors. In some embodiments, a sample of DNA may be amplified for a number of cycles by using a first set of amplification primers that comprise target specific regions and non-target specific tag regions and a first PCR master mix. The tag region can be any sequence, such as a universal tag region, a capture tag region, an amplification tag region, a sequencing tag region, a UMI tag region, and the like. For example, a tag region can be the template for amplification primers utilized in a second or subsequent round of amplification, for example for library preparation. In some embodiments, the methods comprise adding single stranded-binding protein (SSB) to the first amplification products. An aliquot of the first amplified sample can be removed and amplified a second time using a second set of amplification primers that are specific to the tag region, e.g., a universal tag region or an amplification tag region, of the first amplification primers which may comprise of one or more additional tag sequences, such as sequence tags specific for one or more downstream sequencing workflows, and the same or a second PCR master mix. As such, a library of the original DNA sample is ready for sequencing.

An alternative method could comprise the first amplification being performed in a small volume (e.g., 15 ul) and instead of transferring an aliquot to a new location for a second round of amplification, additional reagents to perform a second round of amplification could be added to the tube.

Once the library is created, it can be purified and quantitated. In some examples, purification can be performed by processing the sample through a substrate such as AMPURE XP Beads (Beckman Coulter) which serves to purify the DNA fragments away from reaction components. Another method could be the incorporation of a purification moiety, such as a hapten moiety, into the second set of amplification primers. For example, if a biotin was incorporated into one the primers of the second amplification primer set then the library fragments could be capturing using a streptavidin moiety on a bead for example. Utilizing the capture strategy the libraries could also be normalized and quantitated using Bead Based Normalization (BBN). However, libraries can be purified and quantitated, or pooled and quantitated if multiple reactions are being performed, without the use of BBN. For example, libraries could also be quantitated by gel electrophoretic methods, microfluidics-based automated electrophoresis methods, e.g., using BIOANALYZER, qPCR, spectrophotometric methods, quantitation kits (e.g., PicoGreen™, etc.) and the like as known in the art. Following quantitation, the library can then be sequenced by parallel sequencing.

In some embodiments, the first set of amplification primers used to amplify a target DNA is provided in such a limited concentration that when an aliquot of the first amplification reaction is added to a new tube and the reagents from the second amplification reaction are added there is minimal to undetectable carryover amplification resulting from the first set of amplification primers and a cleanup step between the first amplification reaction and the second amplification reaction is not required. In some examples, the concentration of the amplification primers for a first PCR is, is about, or is less than, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1.0 nM, 1.5 nM, 2.0 nM, 3.0 nM, 4.0 nM, 5.0 nM, 6.0 nM, 7.0 nM, 8.0 nM, 9.0 nm, 10.0 nM, 11.0 nM, 12.0 nM, or a range between any of these values, for example, 0.5 nM to 1.0 nM, 1.0 nM to 12 nM, 0.8 nM to 1.5 nM, etc. In some embodiments, the concentration of amplification primers for a first PCR is about 0.9 nM to about 10 nM.

FIG. 2 shows an exemplary workflow of the presently disclosed methods in one embodiment. A target genomic DNA sequence is amplified using a first set of primers comprising a region that flanks the target sequence and amplification tag regions (which may be the same or different) resulting in amplicons comprising the target sequence and tags on both ends. An aliquot of the amplicons from the first PCR is further amplified using a second set of primers specific to the first tag sequences further comprising sequencing primer sequences (i5 and i7 adapter sequences), thereby generating a library comprising the target DNA sequence flanked by sequences used in parallel sequencing, in this case i5 and i7 sequences are utilized in sequence by synthesis methods popularized by Illumina, Inc.

An example of an alternative workflow for determining a DNA profile from a sample is described in FIG. 3 . In this example, a DNA target is amplified with a first primer pair that comprises sequences that flank the target sequence, non-target tag sequences (the same or different) and further unique molecular identifier sequences or UMIs, which comprise randomized bases. The UMIs can be used, for example, to bioinformatically decrease or eliminate errors that occur during the library preparation processes (e.g., PCR artifacts or misincorporations, etc.). Use of UMIs can be important for DNA profiling, but are of particular importance for use in helping to eliminate errors when samples are sequenced for criminal casework. In this example, the first round of amplification is performed for 2 cycles, which is followed by addition of a single stranded binding protein (SSB) and incubation at 37 degree C. for 15 min following by a 95 degree C./5 min inactivation which effectively quenches further amplification of the first set of amplification primers during the second round of amplification. Although the mechanism is unknown, it is contemplated that adding the SSB irreversibly binds the single stranded first amplification primers and prevents them from participating in subsequent amplification reactions. Following SSB incubation, a second set of primers comprising sequence tags and a second PCR mix is added resulting in the sequencing library.

Nucleic Acid Library

Embodiments disclosed herein provide nucleic acid libraries, which may be used for sequencing. In some embodiments, the nucleic acid libraries disclosed herein may comprise a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprise at least one tandem repeat sequence flanked by a first pair of tag sequences and at least one single nucleotide polymorphism (SNP) sequence flanked by a second pair of tag sequences.

As outlined herein, the size of the nucleic acid molecules may vary greatly using the methods and compositions disclosed herein. It would be appreciated by those skilled in the art that the nucleic acid molecules amplified from a target sequence comprising a tandem repeat (e.g., STR) may have a large size, while the nucleic acid molecules amplified from a target sequence comprising a SNP may have a small size. For example, the nucleic acid molecules may comprise from less than a hundred nucleotides to hundreds or even thousands of nucleotides. Therefore, the size of the nucleic acid molecules may have a range that is between any two values of about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp, about 110 bp, about 120 bp, about 130 bp, about 140 bp, about 150 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1 kb, or more. In some embodiments, the minimal size of the nucleic acid molecules may be a length that is, is about, or is less than, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, or 100 bp. In some embodiments, the maximum size of the nucleic acid molecules may be a length that is, is about, or is more than, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, or 1 kb.

For cluster generation, the library fragments are immobilized on a substrate, for example a slide, which comprises homologous oligonucleotide sequences for capturing and immobilizing the DNA library fragments. The immobilized DNA library fragments are amplified using cluster amplification methodologies as exemplified by the disclosures of U.S. Pat. Nos. 7,985,565 and 7, 115,400, the contents of each of which is incorporated herein by reference in its entirety. The incorporated materials of U.S. Pat. Nos. 7,985,565 and 7,115,400 describe methods of solid-phase nucleic acid amplification which allow amplification products to be immobilized on a solid support in order to form arrays comprised of clusters or “colonies” of immobilized nucleic acid molecules. Each cluster or colony on such an array is formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary polynucleotide strands. The arrays so-formed are generally referred to as “clustered arrays”. The products of solid-phase amplification reactions such as those described in U.S. Pat. Nos. 7,985,565 and 7,115,400 are so-called “bridged” structures formed by annealing of pairs of immobilized polynucleotide strands and immobilized complementary strands, both strands being immobilized on the solid support at the 5′ end, preferably via a covalent attachment. Cluster amplification methodologies are examples of methods wherein an immobilized nucleic acid template is used to produce immobilized amplicons. Other suitable methodologies can also be used to produce immobilized amplicons from immobilized DNA fragments produced according to the methods provided herein. For example one or more clusters or colonies can be formed via solid-phase PCR whether one or both primers of each pair of amplification primers are immobilized. However, the methods described herein are not limited to any particular sequencing preparation methodology or sequencing platform and can be amenable to other parallel sequencing platform preparation methods and associated sequencing platforms.

Primers

Embodiments disclosed herein provide a plurality of primers that specifically hybridize to at least one short target sequence and at least one long target sequence in a nucleic acid sample, wherein amplifying the nucleic acid sample using the plurality of primers in a single multiplex reaction results in at least one short amplification product and at least one long amplification product, wherein each of the plurality of primers comprises one or more tag sequences. Further disclosed herein is a plurality of primers that have the sequences set forth in Tables 1-2.

For multiplex amplification of large target sequence (e.g., STRs, ITRs) and small target sequence (e.g., SNPs), primers are designed that would allow for balanced amplification across all the target types. The methods and compositions disclosed herein may be used to amplify multiple tandem repeat target sequences in a single multiplex reaction. For example, the plurality of primers may specifically hybridize to a number of tandem repeat sequences that is, is about, or is more than 4, 6, 8, 10, 12, 14, 16, 18, 24, 30, 40, 50, 60, 70, 80, 90, 100, or a range between any of the two values, such as 4 to 12, 10 to 24, 30 to 100, etc. In some embodiments, the plurality of primers may specifically hybridize to at least 24 tandem repeat sequences. In some embodiments, the plurality of primers may specifically hybridize to at least 60 tandem repeat sequences. The methods and compositions disclosed herein may be used to amplify multiple SNP target sequences in a single reaction. For example, the plurality of primers may specifically hybridize to a number of SNP sequences that is, is about, or is more than 4, 6, 8, 10, 12, 14, 16, 18, 24, 30, 40, 50, 60, 70, 80, 90, 100, or a range between any of the two values, such as 4 to 12, 10 to 24, 30 to 100, etc. In some embodiments, the plurality of primers may specifically hybridize to at least 30 SNP sequences. In some embodiments, the plurality of primers may specifically hybridize to at least 50 SNP sequences.

It was discovered during experimentation that the short SNP target sequences preferentially amplified over the longer STR target sequences when using primers that were designing following established criteria and wisdom for successful primer design. Further, at least in the sequence by synthesis workflow where clusters are generated and the clusters are themselves sequenced (for example, when following sequence by synthesis (SBS, disclosed herein elsewhere) associated with the Illumina, Inc. sequencers) preferential cluster amplification of the shorter library SNP fragments also occurred. To overcome these two biases, a new strategy was needed for primer design that would allow for balanced amplification between the short SNP target sequences and the long STR target sequences.

One of the strategies included designing primers for STR amplification. With STRs, the repeated sequences are often embedded within larger repeated regions; therefore designing specific primers for STR amplification can be problematic. Further, STRs and their flanking regions are oftentimes AT rich. In one instance, primers were designed to the problematic regions using a design strategy contrary to conventional and well established PCR design criteria. The established criteria for PCR primer design states that, among other criteria, 1) optimal length for primers is 18-22 nucleotides, 2) the Tm should be in the range of 55-58 degrees C., 3) GC content should be around 40-60%, 4) and repeated AT dinucleotide regions should be avoided, with <4 dinucleotide AT repeats being the maximum. Primers were designed that were longer than typical PCR primers, for example 23-35 nucleotides long instead of 18-22 nucleotides, they had low melting temperatures (Tm), for example around 54 degrees C. instead of around 58 degrees C., and the primers were AT rich, three parameters that conventional established PCR criteria teach should be avoided for optimal primer design. In effect, non-optimal primers were designed. Surprisingly, it was discovered that these long, AT rich, low Tm primers actually multiplexed the STRs better than the short, high Tm low AT containing primers. Without being bound to any theory, it is contemplated that the shorter primers that were designed following established PCR design criteria might form dimers that had high melting temperatures and thus formed dimers efficiently under normal PCR conditions, whereas the longer, low Tm primers might form dimers under really low Tm and thus would not be stable for dimer formation, thereby allowing for increased participation of the longer, low Tm primers under normal amplification conditions compared to the short, high Tm primers (e.g., 18-22 nucleotides, Tm of 60 degrees C., 50% GC content).

The longer, low Tm, AT rich primers for STR amplification were then multiplexed with the conventionally designed, high Tm shorter primers that targeted SNPs. However, the multiplex amplification reactions were once again unsuccessful in providing a balanced amplification of both STRs and SNPs in one multiplex reaction. It was contemplated that perhaps applying the unconventional primer design to amplify non-problematic targets, for example to amplify the SNP targets, might yield successful multiplex amplifications. As such, the same criteria used to design non-optimal primers for STRs were applied to primer design for SNPs (long, low Tm, AT rich). Surprisingly, the new designed primers resulted in better balance between amplification of STRs and SNPs in a multiplex reaction.

FIG. 4 shows examples of the interplay between the conventional and unconventional designed primers in a multiplex reaction. In FIG. 4A a multiplex reaction of 10 SNP targets shows expected amplification in the desired range of around 200-350 bp for the library. The primers used to amplify the 10 SNPs in the multiplex were designed to be longer, have lower Tm and be more AT rich that is advised by established PCR primer design criteria. When an 11th primer pair is designed using the established PCR design criteria, that is the primers are short, have high Tm and are not AT rich, and added to the 10 pairs the resulting multiplex shows non-specific amplification of the target DNA As seen in FIGS. 4B and 4D, the addition of an 11th conventionally designed primer pair interferes with the 10 plex of unconventional primer pairs and results in an unsuccessful multiplex amplification of targeted SNPs. However, the addition of an 11th primer pair that is also unconventionally designed following the same criteria as the 10 plex of primer pairs results in the successful amplification of the SNP targets (FIG. 4C).

Accordingly, in some embodiments, each of the plurality of primers has a low melting temperature, e.g., less than 60 degrees C. or about 50 degrees C. to about 60 degrees C., and/or has a length of at least 24 nucleotides, e.g., about 24 nucleotides to about 38 nucleotides. In some embodiments, each of the plurality of primers comprises a homopolymer nucleotide sequence.

In some examples, the unconventionally designed primers comprise sequences that flank the targeted STRs and SNPs and additional non-template sequences. The additional sequences can be, for example tag sequences that serve a purpose during library preparation or sequencing methodologies. For example, a tag sequence can be a capture sequence such as a hapten moiety than can be captured by an immobilized partner moiety for purifying library fragments. An example of a hapten moiety is biotin which can be captured by streptavidin for isolated library fragments from reaction components and the like. A tag sequence could also be an amplification sequence, for example that is complementary to an amplification primer and is used in one or more amplification reactions. FIGS. 2 and 3 show examples of tag sequences that are used in a second round of amplification following a first round of amplification. A tag sequence could also be a sequence tag. FIGS. 2 and 3 also show examples of sequence tags, i5 adapter and i7 adapter are used in sequencing as hybridization, cluster generation and sequencing primers during the sequence by synthesis reactions as described herein. Another example of a tag sequence is a unique molecular identifier, or UMI, as shown in FIG. 3 .

A UMI comprises a random stretch of nucleotides that can be used during sequencing to correct for PCR and sequencing errors, thereby adding an additional layer of error correction to sequencing results. UMIs could be from, for example 3-10 nucleotides long, however the number will depend on the amount of input DNA For example, if 1 ng DNA is used to target around 250 sites, then it is anticipated that approximately 350 copies×250 targets would be needed, so approximately 90,000 different UMIs. If more DNA is utilized, for example 10 ng, then approximately 1 million different UMIs could be needed. All PCR duplicates from the same PCR reaction would have the same UMI sequence, as such the duplicates can be compared and any errors in the sequence such as single base substitutions, deletions, insertions (i.e., stutter in PCR) can be excluded from the sequencing results bioinformatically. Unique molecular identifiers can also be used in analysis for a mixed sample. Mixed samples, for example a female DNA sample that is contaminated with male DNA, can be deconvoluted to report both the female and male DNA contributions using UMI sequences. For example, there could be a total of four different repeated numbers for two mixed DNAs; however there could be less than four if the mixture of two samples shares alleles at a particular locus. These shared alleles can be distinguished and approximate percentages determined using the UMIs for determining the number of different alleles in the initial population of DNA molecules. For example, the initial molecules could be counted and if a minor contributor was present at, for example 5%, then 5% of the UMIs would identify one genotype and 95% would identify a second genotype. After PCR, if one of the alleles (or perhaps more) was biased upon amplification then that 5:95 ratio would not be seen. However, using UMIs a biased ratio could be corrected after PCR duplicates are condensed using UMI detection and correction. This is important when trying to differentiate from a stutter artifact from PCR and a true minor contributor.

A primer of the present methods can comprise one or more tag sequences. The tag sequences can be one or more of primer sequences that are not homologous to the target sequence, but for example can be used as templates for one or more amplification reactions. The tag sequence can be a capture sequence, for example a hapten sequence such as biotin that can be used to purify amplicons away from reaction components. The tag sequences can be sequences such as adaptor sequences that are advantageous for capturing the library amplicons on a substrate for example for bridge amplification in anticipation of sequence by synthesis technologies as described herein. Further, tag sequences can be unique molecular identifier tags of typically between, for example, 3-10 nucleotides comprised of a randomized stretch of nucleotides that can be used for error correction during library preparation and/or sequencing methods.

Additionally, it is advantageous for a multiplexed PCR reaction to contain oligonucleotide primers to substantially all of the targets pooled together into one mix. However, as disclosed herein, the oligonucleotides are uncharacteristically longer than primers designed using traditional parameters. Further addition of tag sequences to the primers, such as the addition of UMIs that append a gene target specific sequence create still longer primer sequences. In some embodiments, glycine betaine (approximately 1.5M) may be added to the plurality of primers. For example, in some embodiments the amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a betaine concentration that is, is about, or is more than, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2M, 3M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M, 10M, or a range between any two of these values, for example, from 500 mM to 2 M, from 1 M to 1.5 M, etc. As such, a primer mix as described herein supplemented with betaine, for example at approximately 1.5M, would be advantageous when practicing methods of the present disclosure. In some embodiments, glycerol may be added to the plurality of primers. For example, m some embodiments the amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a glycerol concentration that is, is about, or is more than, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2M, 3M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M, 10 M, or a range between any two of these values, for example, from 500 mM to 2 M, from 1 M to 1.5 M, etc. As such, a primer mix as described herein supplemented with glycerol, for example at approximately 1.5M, would be advantageous when practicing methods of the present disclosure.

In some embodiments, buffers associated with unconventional primer design used in amplification methods of the present disclosure may also be modified. For example, in some embodiments the salt concentrations, such as KCl, LiCl, NaCl, or a combination thereof, of the amplification buffer are increased compared to the salt concentration of an amplification buffer used in conjunction with conventionally designed primers. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a KCl concentration that is, is about, or is more than, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM, or a range between any two of these values, for example, from 60 mM to 200 mM, from 100 mM to 250 mM, etc. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a KCl concentration that is about 145 mM. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a LiCl concentration that is, is about, or is more than, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM, or a range between any two of these values, for example, from 60 mM to 200 mM, from 100 mM to 250 mM, etc. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a LiCl concentration that is about 145 mM. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a NaCl concentration that is, is about, or is more than, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM, or a range between any two of these values, for example, from 60 mM to 200 mM, from 100 mM to 250 mM, etc. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a NaCl concentration that is about 145 mM.

In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein may comprise MgSO₄, MgCl₂, or a combination thereof.

Embodiments disclosed herein provide kits comprising at least one container means, wherein the at least one container means comprises a plurality of primers as disclosed herein. In some embodiments, the container means may be a tube, a well, a microtiter plate, etc. In some embodiments, the plurality of primers may specifically hybridize to a number of tandem repeat sequences that is, is about, or is more than 4, 6, 8, 10, 12, 14, 16, 18, 24, 30, 40, 50, 60, 70, 80, 90, 100, or a range between any of the two values, such as 4 to 12, 10 to 24, 30 to 100, etc. In some embodiments, the plurality of primers may specifically hybridize to at least 24 tandem repeat sequences. In some embodiments, the plurality of primers may specifically hybridize to at least 60 tandem repeat sequences. The methods and compositions disclosed herein may be used to amplify multiple SNP target sequences in a single reaction. For example, the plurality of primers may specifically hybridize to a number of SNP sequences that is, is about, or is more than 4, 6, 8, 10, 12, 14, 16, 18, 24, 30, 40, 50, 60, 70, 80, 90, 100, or a range between any of the two values, such as 4 to 12, 10 to 24, 30 to 100, etc. In some embodiments, the plurality of primers may specifically hybridize to at least 30 SNP sequences. In some embodiments, the plurality of primers may specifically hybridize to at least 50 SNP sequences.

In some embodiments, the at least one container means comprise an amplification buffer. In some embodiments, buffers associated with unconventional primer design used in amplification methods of the present disclosure may also be modified. For example, in some embodiments the salt concentrations, such as KCl, LiCl, NaCl, or a combination thereof, of the amplification buffer are increased compared to the salt concentration of an amplification buffer used in conjunction with conventionally designed primers. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a KCl, NaCl or LiCl concentration that is, is about, or is more than, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM, or a range between any two of these values, for example, from 60 mM to 200 mM, from 100 mM to 250 mM, etc. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a KCl, NaCl or LiCl concentration that is about 145 mM.

In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein may comprise MgSO₄, MgCl₂, or a combination thereof.

Sequencing Methods

The present methods are not limited to any particular sequencing platform, however are being exemplified here in regards to SBS, or sequence by synthesis, type of parallel sequencing. Particularly applicable techniques are those wherein nucleic acids are attached at fixed locations in an array such that their relative positions do not change and wherein the array is repeatedly imaged. Examples in which images are obtained in different color channels, for example, coinciding with different labels used to distinguish one nucleotide base type from another are particularly applicable.

SBS techniques generally involve the enzymatic extension of a nascent nucleic acid strand through the iterative addition of nucleotides against a template strand. In traditional methods of SBS, a single nucleotide monomer may be provided to a target nucleotide in the presence of a polymerase in each delivery. However, in the methods described herein, more than one type of nucleotide monomer can be provided to a target nucleic acid in the presence of a polymerase in a delivery.

SBS techniques can utilize nucleotide monomers that have a label moiety or those that lack a label moiety. Accordingly, incorporation events can be detected based on a characteristic of the label, such as fluorescence of the label; a characteristic of the nucleotide monomer such as molecular weight or charge; a byproduct of incorporation of the nucleotide, such as release of pyrophosphate; or the like. In some examples where two or more different nucleotides are present in a sequencing reagent, the different nucleotides can be distinguishable from each other, or alternatively, the two or more different labels can be the indistinguishable under the detection techniques being used. For example, the different nucleotides present in a sequencing reagent can have different labels and they can be distinguished using appropriate optics as exemplified by the sequencing methods developed by Solexa (now Illumina, Inc.).

Some examples include pyrosequencing techniques. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) “Real-time DNA sequencing using detection of pyrophosphate release.” Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) “Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P. (1998) “A sequencing method based on real-time pyrophosphate.” Science 281(5375), 363; U.S. Pat. Nos. 6,210,891; 6,258,568 and 6,274,320, the disclosures of which are incorporated herein by reference in their entireties).

In pyrosequencing, released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated is detected via luciferase-produced photons. The nucleic acids to be sequenced can be attached to features in an array and the array can be imaged to capture the chemiluminescent signals that are produced due to incorporation of a nucleotides at the features of the array. An image can be obtained after the array is treated with a particular nucleotide type (e.g. A, T, C or G). Images obtained after addition of each nucleotide type will differ with regard to which features in the array are detected. These differences in the image reflect the different sequence content of the features on the array. However, the relative locations of each feature will remain unchanged in the images. The images can be stored, processed and analyzed using the methods set forth herein. For example, images obtained after treatment of the array with each different nucleotide type can be handled in the same way as exemplified herein for images obtained from different detection channels for reversible terminator-based sequencing methods.

In another example of SBS, cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in WO 04/018497 and U.S. Pat. No. 7,057,026, the disclosures of which are incorporated herein by reference. This approach is being commercialized by Solexa (now Illumina Inc.), and is also described in WO 91/06678 and WO 07/123,744, each of which is incorporated herein by reference. The availability of fluorescently-labeled terminators in which both the termination can be reversed and the fluorescent label cleaved facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides. Additional exemplary SBS systems and methods which can be utilized with the methods and systems described herein are described in U.S. Patent Application Publication No. 2007/0166705, U.S. Patent Application Publication No. 2006/0188901, U.S. Pat. No. 7,057,026, U.S. Patent Application Publication No. 2006/0240439, U.S. Patent Application Publication No. 2006/0281109, PCT Publication No. WO 05/065814, U.S. Patent Application Publication No. 2005/0100900, PCT Publication No. WO 06/064199, PCT Publication No. WO 07/010,251, U.S. Patent Application Publication No. 2012/0270305 and U.S. Patent Application Publication No. 2013/0260372, the disclosures of which are incorporated herein by reference in their entireties.

Some examples can utilize detection of four different nucleotides using fewer than four different labels. For example, SBS can be performed utilizing methods and systems described in the incorporated materials of U.S. Patent Application Publication No. 2013/0079232. As a first example, a pair of nucleotide types can be detected at the same wavelength, but distinguished based on a difference in intensity for one member of the pair compared to the other, or based on a change to one member of the pair (e.g. via chemical modification, photochemical modification or physical modification) that causes apparent signal to appear or disappear compared to the signal detected for the other member of the pair. As a second example, three of four different nucleotide types can be detected under particular conditions while a fourth nucleotide type lacks a label that is detectable under those conditions, or is minimally detected under those conditions (e.g., minimal detection due to background fluorescence, etc.). Incorporation of the first three nucleotide types into a nucleic acid can be determined based on presence of their respective signals and incorporation of the fourth nucleotide type into the nucleic acid can be determined based on absence or minimal detection of any signal. As a third example, one nucleotide type can include label(s) that are detected in two different channels, whereas other nucleotide types are detected in no more than one of the channels. The aforementioned three exemplary configurations are not considered mutually exclusive and can be used in various combinations. An exemplary embodiment that combines all three examples, is a fluorescent-based SBS method that uses a first nucleotide type that is detected in a first channel (e.g. dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type that is detected in a second channel (e.g. dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type that is detected in both the first and the second channel (e.g. dTTP having at least one label that is detected in both channels when excited by the first and/or second excitation wavelength) and a fourth nucleotide type that lacks a label that is not, or minimally, detected in either channel (e.g. dGTP having no label).

Further, as described in the incorporated materials of U.S. Patent Application Publication No. 2013/0079232, sequencing data can be obtained using a single channel. In such so-called one-dye sequencing approaches, the first nucleotide type is labeled but the label is removed after the first image is generated, and the second nucleotide type is labeled only after a first image is generated. The third nucleotide type retains its label in both the first and second images, and the fourth nucleotide type remains unlabeled in both images.

Some examples can utilize sequencing by ligation techniques. Such techniques utilize DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. The oligonucleotides typically have different labels that are correlated with the identity of a particular nucleotide in a sequence to which the oligonucleotides hybridize. As with other SBS methods, images can be obtained following treatment of an array of nucleic acid features with the labeled sequencing reagents. Each image will show nucleic acid features that have incorporated labels of a particular type. Different features will be present or absent in the different images due the different sequence content of each feature, but the relative position of the features will remain unchanged in the images. Images obtained from ligation-based sequencing methods can be stored, processed and analyzed as set forth herein. Exemplary SBS systems and methods which can be utilized with the methods and systems described herein are described in U.S. Pat. Nos. 6,969,488, 172,218, and 6,306,597, the disclosures of which are incorporated herein by reference in their entireties.

Some examples can utilize nanopore sequencing (Deamer, D. W. & Akeson, M. “Nanopores and nucleic acids: prospects for ultrarapid sequencing.” Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, “Characterization of nucleic acids by nanopore analysis”. Ace. Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J. A Golovchenko, “DNA molecules and configurations in a solid-state nanopore microscope” Nat. Mater. 2:611-615 (2003), the disclosures of which are incorporated herein by reference in their entireties). In such embodiments, the target nucleic acid passes through a nanopore. The nanopore can be a synthetic pore or biological membrane protein, such as a-hemolysin. As the target nucleic acid passes through the nanopore, each base-pair can be identified by measuring fluctuations in the electrical conductance of the pore. (U.S. Pat. No. 7,001,792; Soni, G. V. & Meller, “A Progress toward ultrafast DNA sequencing using solid-state nanopores.” Clin. Chem. 53, 1996-2001 (2007); Healy, K. “Nanopore-based single-molecule DNA analysis.” Nanomed. 2, 459-481 (2007); Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. “A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution.” J. Am. Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference in their entireties). Data obtained from nanopore sequencing can be stored, processed and analyzed as set forth herein. In particular, the data can be treated as an image in accordance with the exemplary treatment of optical images and other images that is set forth herein.

Some examples can utilize methods involving the real-time monitoring of DNA polymerase activity. Nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and y-phosphate-labeled nucleotides as described, for example, in U.S. Pat. Nos. 7,329,492 and 7,211,414 (each of which is incorporated herein by reference) or nucleotide incorporations can be detected with zero-mode waveguides as described, for example, in U.S. Pat. No. 7,315,019 (which is incorporated herein by reference) and using fluorescent nucleotide analogs and engineered polymerases as described, for example, in U.S. Pat. No. 7,405,281 and U.S. Patent Application Publication No. 2008/0108082 (each of which is incorporated herein by reference). The illumination can be restricted to a zeptoliter-scale volume around a surface-tethered polymerase such that incorporation of fluorescently labeled nucleotides can be observed with low background (Levene, M. J. et al. “Zero-mode waveguides for single-molecule analysis at high concentrations.” Science 299, 682-686 (2003); Lundquist, P. M. et al. “Parallel confocal detection of single molecules in real time.” Opt. Lett. 33, 1026-1028 (2008); Korlach, J. et al. “Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nano structures.” Proc. Natl. Acad. Sci. USA I 05, 1176-1181(2008), the disclosures of which are incorporated herein by reference in their entireties). Images obtained from such methods can be stored, processed and analyzed as set forth herein.

Some SBS embodiments include detection of a proton released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, Conn., a Life Technologies subsidiary) or sequencing methods and systems described in US 2009/0026082 A1; US 2009/0127589 A1; US 2010/0137143 A1; or US 2010/0282617 A1, each of which is incorporated herein by reference. Methods set forth herein for amplifying target nucleic acids using kinetic exclusion can be readily applied to substrates used for detecting protons. More specifically, methods set forth herein can be used to produce clonal populations of amplicons that are used to detect protons.

The above SBS methods can be advantageously carried out in multiplex formats such that multiple different target nucleic acids are manipulated simultaneously. In particular embodiments, different target nucleic acids can be treated in a common reaction vessel or on a surface of a particular substrate. This allows convenient delivery of sequencing reagents, removal of unreacted reagents and detection of incorporation events in a multiplex manner. In embodiments using surface-bound target nucleic acids, the target nucleic acids can be in an array format. In an array format, the target nucleic acids can be typically bound to a surface in a spatially distinguishable manner. The target nucleic acids can be bound by direct covalent attachment, attachment to a bead or other particle or binding to a polymerase or other molecule that is attached to the surface. The array can include a single copy of a target nucleic acid at each site (also referred to as a feature) or multiple copies having the same sequence can be present at each site or feature. Multiple copies can be produced by amplification methods such as, bridge amplification or emulsion PCR as described in further detail below.

The methods of the present disclosure utilize the Illumina, Inc. technology for sequencing the DNA profile libraries created by practicing the methods described herein. The MISEQ System (a sequencing instruments was used for clustering and sequencing for the examples described herein. However, as previously stated and as understood by a skilled artisan, the present methods are not limited by the type of sequencing platform used.

EXAMPLES

The following examples disclose several methods and materials for DNA profiling. These methods and materials can be modified while maintaining the spirit and scope of the invention. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the methods disclosed here. Consequently, it is not intended that these methods or materials be limited to the specific examples disclosed herein, but that it cover all modifications and alternatives that fall within the scope and spirit of the present disclosure.

Example 1 Unconventional Primer Design

The computer design program DesignStudio from Illumina, Inc. (San Diego, Calif.) was modified and used for primer design. A skilled artisan would of course understand that alternative primer design programs such as Primer3 can also be used and the default parameters reset to mimic the intent of the modified parameters for primer design. The settings are typically reset in the config.xml file that comes with the software, however this may differ when using different software and consulting the specific materials for accessing default parameters for each software is typical practice. The following parameters can be reset in the primer design software:

-   -   1) The desired minimum length amplicon reset to >60<     -   2) The desired maximum length amplicon reset to >120<     -   3) The tight candidate spacing reset to >3< (default is 30 bp)     -   4) The % GC Max probe reset to >60< to allow for increased         number of AT rich repeat stretches     -   5) The mean Tm reset to >57< (default 59 C) to lower the mean Tm     -   6) The maximum Tm reset to >60< (default 71)     -   7) The minimum Tm reset to >51< (default 55)     -   8) The mean probe length reset to >28< (default 27)     -   9) The maximum probe length reset to >38< (default 30)     -   10) The minimum probe length reset to >25< (default 22)

For designing the SNP primers, the range to target for the 3′ end of the primer was set to “small” to keep the primers around 1 bp away for the targeted SNP. Once all the parameters are reset, the primer design program can be run on the sequence to determine the primer pair candidates that fall under the new parameters. For example, a user of the software can generate a targets list that tells the software where to look in the genome for designing the primers. In the present example, the targeted regions were copy and pasted into the graphic user interface application which the DesignStudio software used to orient and target primer design. Once the targeted regions were input into the program, the program directed to Create a Design File to start the tool and create the primer designs. In the present example, the main output was a .txt file that included the primer sequences and/or some of the regions contained failures and were “undesignable”, at which point the targeted sequences needed to be redefined and rerun. The software used in this experiment provided the designed primers that were mapped onto the sequence that was specified as the targeted region. Following the reset parameters, primers were designed that did not follow the conventional criteria for primer design for amplification; however which allowed for the multiplex amplification of long STRs and short SNPs.

Examples of designed STR targeted primers advantageous in methods disclosed herein include those listed in Table 1. Examples of SNP targeted primers advantageous in methods disclosed herein include those listed in Table 2.

TABLE 1 STR targeted primer without tags and amplicon sizes SEQ ID AMPLICON NO STR LOCUS PRIMER EXAMPLES OF STR PRIMERS WITHOUT TAGS SIZE 1 AmeiPP F T CCCTGGGCTCTGTAAAGAA 106, 112 2 AmeiPP R 5m ATCAGAGCTTAAACTGGGAAGCTG 3 C5F1PO F1 T ACAGTAACTGCCTTCATAGATAG 117 4 C5F1PO R1 5m GTGTCAGACCCTGTTCTAAGTA 5 055818 F2 T TGATTTTCCTCTTTGGTATCCTTATGTAAT 112 6 055818 R2 5m ACAACATTTGTATCTTTATCTGTATCCT 7 0851179 F1 T TTTGTATTTCATGTGTACATTCGTATC 110 8 0851179 R1 5m ACCTATCCTGTAGATTATTTTCACTGTG 9 018551 F1 T CTCTGAGTGACAAATTGAGACCTT 184 10 018551 R1 T TTAACTTCTCTGGTGTGTGGAGATG 11 0195433 F1 T TTTGGTGCACCCATTACCCG 188 12 0195433 R1 5m AGGAGGTTGAGGCTGCAAAA 13 075820 F2 T CACCAAATATTGGTAATTAAATGTTTACTATAGAC 167 14 075820 R2 5m TAAAGGGTATGATAGAACACTTGTC 15 0165539 F2 T CAAAGGCAGATCCCAAGCTCT 160 16 0165539 R2 5m TGTGTGTGCATCTGTAAGCAT 17 0351358 F2 T TGGTGTGTATTCCCTGTGCC 170 18 0351358 R2 5m GCAGTCCAATCTGGGTGACA 19 01051248 F1 T CCAATCTGGTCACAAACATATTAATGAA 148 20 01051248 R1 5m TTTCCCTTGTCTTGTTATTAAAGGAAC 21 TH01 F1 T TTCCCATTGGCCTGTTCCTC 112 22 TH01 R1 5m CTGTACACAGGGCTTCCGAG 23 FGA F2 T GCTGAGTGATTTGTCTGTAATTG 188 24 FGA R2 5m GAACTCACAGATTAAACTGTAACCAAAATAAAATTAG 25 061043 F1 T CAATAGTGTGCAAGGATGGGTG 175 26 061043 R1 5m TCTGTGGTTCTCCAGCTTAC 27 TPOX F1 T CTTAGGGAACCCTCACTGAATG 77 28 TPOX R1 5m GTCCTTGTCAGCGTTTATTTGC 29 0135317 F2 T TTGGGTTGAGCCATAGGCAG 162 30 0135317 R2 5m GCATCCGTGACTCTCTGGAC 31 021511 F1 T GTTATGGGACTTTTCTCAGTCTCCAT 226 32 D21511 R3 5m GAGACTAATAGGAGGTAGATAGACTGG 33 D125391 Fl T GAGACTGTATTAGTAAGGCTTCTC 253 34 D125391 R2 5m CCTGGACTGAGCCATGCTCC 35 D151656 F2 T CAGTCCTGTGTTAGTCAGGATTC 173 36 D151656 Rl 5m TCAAGGGTCAACTGTGTGATGT 37 D951122 F3 T CTTCTGAAAGCTTCTAGTTTACCT 120 38 D951122 R2 5m TTGCTTATTTGTGGGGGTATTTCA 39 PentaE Fl T AAGAATTCTCTTATTTGGGTTATTAATTG 362 40 PentaE Rl 5m AAATTGTGGACAGGTGCGGT 41 D1751301 F2 T CCATGTAAAAATACATGCATGTGTTTATTTATAC 142 42 D1751301 R2 5m TGATTAAAAAGAATGAAGGTAAAAATGTGTATAAC 43 D25441 F2 T CCAAATGTTTATGATTAATCTTTTAAATTGGAGC 160 44 D25441 R3 5m GTAACAAGGGCTACAGGAATCATGAG 45 D452408_F3_T TCATCCACTGAAATGACTGAAAAATAG 102 46 D452408 R9 5m AGGTACATAACAGTTCAATAGAAAG 47 D251338 F2 T GAGTTATTCAGTAAGTTAAAGGATTGCAG 162 48 D251338 R2 5m GGGAGCCAGTGGATTTGGAAACAG 49 PentaD F3 T GCATGGTGAGGCTGAAGTAG 268 50 PentaD Rl 5m CTAACCTATGGTCATAACGATTTTT 51 vWA F3 T GATGATAAGAATAATCAGTATGTGACTTGG 160 52 vWA R3 5m ATAGGTTAGATAGAGATAGGACAGATGATA 53 5E33 Fl T CCCTACCGCTATAGTAACTTGC 380 54 5E33 R2 5m CACGTCTGTAATTCCAGCTCCTA 55 D205482 F3 T GGAAGCGTGTACTAGAGTTCTTCAG 145 56 D205482 R2 5m GGACAGCCTCCATATCCACATG 57 DX510074_Fl_T TTCCTACTGCCCCACCTTTATTG 212 58 DX510074 Rl sm TTTATGGTCTCAGTGCCCCTCAGA 59 DX510103 Fl sm TCATAATCACATATCACATGAGC 177 60 DX510103 Rl T AAACAGAACCAGGGGAATGAA 61 DX510135_Fl_T TGAAACTAAAGTCAAATGGGGCTAC 268 62 DX510135_Rl_sm TAAGGGGTGACACCTCTCTGGATA 63 DX58377_F2_sm CCCAGCCTACATCTACCACTTCATG 276 64 DX58377 R2 T CTAATGTTCGTATGGACCTTTGGAAAGC 65 DX57423 Fl sm GTCTCCAGTACCCAGCTAGCTTAG 191 66 DX57423 Rl T TCTCCCAACCTGCCCTTTATCA 67 DX58378 Fl sm TTTGGGCTGACACAGTGGCT 442 68 DX58378 Rl T TTGATCAACACAGGAGGTTTGACC 69 HPRTB_Fl_sm TATACCACTTTGATGTTGACACTAGTTTAC 213 70 HPRTB Rl T CCTGTCTATGGTCTCGATTCAAT 71 DX510148_F3_sm TGCATGACAGAGGGAGATTCT 256 72 DX510148 R3 T AGAGGGGAAATAGTAGAATGAGGATG 73 DX57132_F3_sm GCCAAACTCTATTAGTCAACGTTC 204 74 DX57132_R4_T CTGGTTCTCTAGCTCACATACAGT 75 DYF38751ab F2 T TTTACCCCTAACAAGAAAAAAAGAAGAA 227, 231 76 DYF38751ab R2 5m CAGTGTGAGAAGTGTGAGAAGTGC 77 DY5385a b Fl T GACACCATGCCAAACAACAAC 260, 248 78 DY5385a b Rl 5m ATCTATCTATTCCAATTACATAGTCC 79 DY53891 II F3 T TCATTATACCTACTTCTGTATCCAACTCTC 183, 303 80 DY53891 II R3 5m GGAACACAATTATCCCTGAGTAGCAG 81 DY5390 F2 T GGTAGCATAATAGAAATTTTATGAGTGGG 318 82 DY5390 R2 5m GAAGACAGACTTCAATATCACAGAACATCG 83 DY5391 Fl T GTGTATCTATTCATTCAATCATACACCC 143 84 DY5391 Rl 5m CTCCCTGGTTGCAAGCAATTGCC 85 DY5438 Fl T CCAAAATTAGTGGGGAATAGTTGAAC 149 86 DY5438 R2 5m GTCGAGATCACACCATTGCATTTC 87 DYS439 F1 T GCCTGGCTTGGAATTCTTTTACCC 195 88 DYS439 R1 Sm TTTAAGTCTTTAATCTATCTTGAATTAATAGATTC 89 DYS481 F1 T CTTTAAGAGGAGTCTGCTAAAAGGAATG 144 90 DYS481 R3 Sm TCACCAGAAGGTTGCAAGAC 91 DYS505 F1 T TCTGGCGAAGTAACCCAAAC 174 92 DYS505 R1 Sm TCGAGTCAGTTCACCAGAAGG 93 DYS522 F2 T GGAACCAGTGAGAGCCG 306 94 DYS522 R2 Sm CTCAGAGTGCTGAACCCAG 95 DYS533 F2 T GTATTTATTCATGATCAGTTCTTAACTCAACC 206 96 DYS533 R2 Sm CTACCTAATATTTATCTATATCATTCTAATTATGTCTCT 97 DYS549 F1 T CTCTAAAGGTTTTTTTTGGTGGCATAAG 222 98 DYS549 R1 Sm GATTAATACAACAAAAATTTGGTAATCTGAAA 99 DYS570 F1 T CAACCTAAGCTGAAATGCAGATATTC 170 100 DYS570 R1 Sm GTTATGAAACGTAAAATGAATGATGACTAG 101 DYS576 F2 T GCAGTCTCATTTCCTGGAGATGAAGG 191 102 DYS576 R1 Sm CTTGGGCTGAGGAGTTCAATC 103 DYS612 F2 T GCCAGTAAGAATAAAATTACAGCATGAAG 287 104 DYS612 R2 Sm GAATAATCTACCAGCAACAATGGCT 105 DYS635 F4 T TGCCCAATGGAATGCTCTCT 274 106 DYS635 R2 Sm GCTCCATCTCAAACAACAAAAACACAAAAAATG 107 DYS643 F2 T GGGTCATTGAACCTCATGCTCTG 170 108 DYS643 R1 Sm CCCCCCAAAATTCTACTGAAGTAAA 109 Y GATAH4 F2 T TAACAGGATAAATCACCTATCTATGTAT 175 110 Y GATAH4 R2 Sm GCTGAGGAGAATTTCCAAATTTA

TABLE 2 SNP targeted primers SEQ ID EXAMPLES OF SNP  NO SNP PRIMER PRIMERS WITHOUT TAGS 111 rs10092491 iSNPI T F2 CCCGCAAACTAACTAGGATAAATCTCTA 112 rs1015250 iSNPI T F CGACATGGGAAATGTCAGATCATAAGAC 113 rs1024116 iSNPI T F2 CCAGGGAGTGAAAAATCCTTTTATCATC 114 rs1028528 iSNPI T F2 GAGGATGAAGGTTAGAGCCAGACCT 115 rs1029047 iSNPI T F2 TGTGGAATAAACTGAAGGCTAAAGAAAA 116 rs1031825 iSNPI T F2 CAAGCCCTATGCCAAGGATATAACAATG 117 rs10488710 iSNPI T F GAGGTTTTACTGTATTAGGAGTTCCCAC 118 rs10495407 iSNPI T F CAGATGTGAGATGATAATTTCGTTCTCC 119 rs1058083 iSNPI T F TTGTTCTTCTCCATCCCATTTCACCC 120 rs10773760 iSNPI T F CTTGTACATTCCCTTATCTGCTATGTGG 121 rs1294331 iSNPI T F2 CTCTCTTTGGAGTTTTATGTGTTGCTAC 122 rs12997453 iSNPI T F CTCTGATGATGTGCAAGAAAGGTAGGTA 123 rs13182883 iSNPI T F TCAGACTATGTTTTAAGGAGACTATGAGG 124 rs13218440 iSNPI T F CTAAGTATCTACCAATGTGCTACGTACC 125 rs1335873 iSNPI T F CACGTGGATGATATGGTTTCTCAAGG 126 rs1336071 iSNPI T F2 AGCACCTATATATTATACCTGAAAGCAT 127 rs1355366 iSNPI T F CCCATGATTTTCTTGTGGTGAGAATTTC 128 rs1357617 iSNPI T F CACCCTCTGTACTTTAATTTGACTTCCC 129 rs1382387 iSNPI T F GTTTTTCTTCATTCCCATGTTGTGTAC 130 rs1413212 iSNPI T F CACTCTTCTGAATCCTGGTCAACAAC 131 rs1454361 iSNPI T F CAAGTTATATCATAGAGTCTACGACCCC 132 rs1463729 iSNPI T F CTGCAACTATCAGTCTCTGCCCTTATTC 133 rs1493232 iSNPI T F GATGTGTCTCAAACTGTTTATTGTGAGG 134 rs1498553 iSNPI T F GAACTCATTTATCCAGAGACCTGTTCTC 135 rs1523537 iSNPI T F CATAATACAACCTGTCTTTGGAGTTACT 136 rs1528460 iSNPI T F GTGACCAGTAGTTCTATGAGCAAGTATG 137 rs159606 iSNPI T F CCACATTGTATGGTTTTTAGGCACCATG 138 rs1736442 iSNPI T F CTAATAAGTGGGACAGTTAAGAGAAGGC 139 rs1821380 iSNPI T F CAAGACAAGCGATTGAAAGAAGTGGAT 140 rs1886510 iSNPI T F CCTTGTCAATCTTTCTACCAGAGGGTAA 141 rs1979255 iSNPI T F GAATCATAGCTTGTGTTGGTCAGGG 142 rs2016276 iSNPI T F GAATTACAAGTATTTGCATCCCAGCCT 143 rs2040411 iSNPI T F GACCAACTTGGCTTTAACAGATGCAAAT 144 rs2046361 iSNPI T F2 TCCTTACCTTTAAGACTTTTCCTATTTG 145 rs2056277 iSNPI T F2 CATTATCTCGTCATACTTCCCTGTCTTG 146 rs2076848 iSNPI T F GCATCAAATTCACCAGTGAAATTATTGA 147 rs2107612 iSNPI T F ATGAGTACATTATTCAACTGTTTTGGAG 148 rs2111980 iSNPI T F CAGCCATGTTGTAAACATTTTTACGGTC 149 rs214955 iSNPI T F GCACATTCTAAGAACTGGTGATTCTATC 150 rs221956 iSNPI T F GCTAGAAAAAGCTGAGATAGCTGTGAAG 151 rs2342747 iSNPI T F CCTTGAAGCTCATTCTTTGTTGTCCC 152 rs2399332 iSNPI T F CTGGACACCAGACCAAAAACAAATAACC 153 rs251934 iSNPI T F GTAATTAGAGGGCAGTGAGGCTTTTAA 154 rs279844 iSNPI T F CTCCAGAAGCTACTGGGATATTAATTAG 155 rs2830795 iSNPI T F TGAGCCAAATCAGCAATATAATAGGACT 156 rs2831700 iSNPI T F CCTAGAACCACAATTATCTGTCTTTGGC 157 rs2920816 iSNPI T F2 CCATTGATTCTCTACAGTTCTGCAGGTA 158 rs321198 iSNPI T F CTCCACACTTTATACAGGTGAAATCTGA 159 rs338882 iSNPI T F CATTTTTCTCTCCTTCTGTCTCACCTTC 160 rs354439 iSNPI T F GCTTCTCTTTCCCTTATGTATCTCTCTC 161 rs3780962 iSNPI T F GGCTTTTGAAGAAAAACACTAACCTGTC 162 rs430046 iSNPI T F CACCTATGGGCTCTTCTTATTTCTCC 163 rs4364205 iSNPI T F CATTTGATAGCCATTTGGGTTGTTTCCA 164 rs445251 iSNPI T F CCATCACACTATCCTGACATGAACAAAT 165 rs4606077 iSNPI T F GAAGATTTGCATCCCAGTGAAAGCAC 166 rs560681 iSNPI T F GCACTTCATAAAGAATCAGTCAGGATGC 167 rs6444724 iSNPI T F GGAGAATCAGGAAATAGTCACTTCCTAC 168 rs6811238 iSNPI T F CATTTGACCTTCTAGCCAAATGAAGTAC 169 rs7041158 iSNPI T F GGAATTTCTGAGAATAACATTGCCTCTC 170 rs717302 iSNPI T F CATATGTTGGGGGAGCTAAACCTAATGA 171 rs719366 iSNPI T F CACTGTGACCACAGCATCTTTTAACTC 172 rs722098 iSNPI T F2 GGGTAAAGAAATATTCAGCACATCCAAA 173 rs722290 iSNPI T F GAGTATCCCTTATCTAAAATGCTGGTCC 174 rs727811 iSNPI T F CTTTTTCTCTTACCGGAACTTCAACGAC 175 rs729172 iSNPI T F CCTCATTAATATGACCAAGGCTCCTCTG 176 rs733164 iSNPI T F TGACTCTAATTGGGGATGTGGTAATTAG 177 rs735155 iSNPI T F GACCTAACCTGGAGAAAACCGGAGA 178 rs740598 iSNPI T F GTTTCTCTTCTCTGAACCTTTGTCTCAG 179 rs740910 iSNPI T F GCAAACACACAAAGATAGGTTCGAGTTT 180 rs763869 iSNPI T F CATATCAAGTGCTTTCTGTTGACATTTG 181 rs8037429 iSNPI T F CTGAAAAGTGCTACGTAAGAGGTCATTG 182 rs8078417 iSNPI T F CATCTGAGTGTGAGAAGAGCCTCAA 183 rs826472 iSNPI T F2 CCCAGCAAAAACTTCTTTTCTCCAGTAA 184 rs873196 iSNPI T F GCTAGGAAAGTTTTCTCTCTGGTTCACA 185 rs876724 iSNPI T F GAATATCTATGAGCAGGCAGTTAGCAG 186 rs891700 iSNPI T F2 CTAATCAGTGTCACTATGTGTGAGCTAT 187 rs901398 iSNPI T F CATCATACAGACTCAAGGAGCTTAGCTG 188 rs907100 iSNPI T F CTTTCCAAGCCTTGGAAAACACAGAAAA 189 rs914165 iSNPI T F GTACCTTATAAATCACGGAGTGCAGAC 190 rs917118 iSNPI T F CAAGTGGTAAGAGATGACTGAGGTCAA 191 rs938283 i5NPI T F CTTCTTCTCTTAGAAGGACACTGGTCAG 192 rs964681 i5NPI T F GTTATGGAGGATTGGTAAGAACCAGAG 193 rs987640 i5NPI T F GAGCTGTTTAAGGGTAAAGGGGTAGTTA 194 rs9905977 i5NPI T F GCAGACAAAACCATGACAATGATCTTAG 195 rs993934 i5NPI T F CCCATGATGAAACAGTTTGCACTAAATG 196 rs9951171 i5NPI T F CTCAATTTTCTTGTCCCTGCTTTCATG 197 rs10092491 i5NPI 5 R2 TTAGAAATTCCAGATAGAGCTAAAACTG 198 rs1015250 i5NPI 5 R GTTAGGAAAAGAACCCAGGTGTTTT 199 rs1024116 i5NPI 5 R2 GCAAAAGTAAATACAAAGGCATACTTT 200 rs1028528 i5NPI 5 R2 CAATGCAAAAGAAAGGTCCTTACTCGAC 201 rs1029047 i5NPI 5 R2 CATTTCTAAACTCTAAAACAAACATTTG 202 rs1031825 i5NPI 5 R2 GGTCCTTAACCTATTAAATTTTAATGAG 203 rs10488710 i5NPI 5 R GACTTTCAATTTATGTCAGCATTTAAAA 204 rs10495407 i5NPI 5 R CCTCTTGGTTGCATTGGATTCTCATTG 205 rs1058083 i5NPI 5 R TCTCCATGAAACTTGGGTTAATTTTGC 206 rs10773760 i5NPI 5 R TGTCTGGAAGTTCGTCAAATTGCAG 207 rs1294331 i5NPI 5 R2 GTAGCATAAAACATTCCAAAAATTCAAT 208 rs12997453 i5NPI 5 R TGCTTTAAAGATACAGGTTATCTGTATTAC 209 rs13182883 i5NPI 5 R CTCTCCGTTACTTTCTTCCTGCCTTT 210 rs13218440 i5NPI 5 R GATCCTGAGATTCACCTCTAGTCCCT 211 rs1335873 i5NPI 5 R CCGTACCAGGTACCTAGCTATGTACT 212 rs1336071 i5NPI 5 R2 CTTTCTGTTTTGTCCATCTGAAATTCT 213 rs1355366 i5NPI 5 R CAAAGTTAAGTATCACCATCCAGCTGG 214 rs1357617 i5NPI 5 R ATAGGGATAGCTGATAAGAAACATGACC 215 rs1382387 i5NPI 5 R CTTAATAAGACGCTGCATCTGCCCA 216 rs1413212 i5NPI 5 R TCCAGGAGACATTTGTTCATATAAGTGA 217 rs1454361 i5NPI 5 R AGACACTTTTCAGTATCCATTTAGAAAC 218 rs1463729 i5NPI 5 R GTTTCACATGTGCATGCTTTTGGGT 219 rs1493232 i5NPI 5 R CCAAAGCTATTCTCTCTTTTGGGTGC 220 rs1498553 i5NPI 5 R GAAAGTTCACTTCAGATGTTCAAAGCC 221 rs1523537 i5NPI 5 R GGGTTTCAGTCTGCAACAAGATCTTG 222 rs1528460 i5NPI 5 R TGGAGATCAATATTTAGCCTTAACATAT 223 rs159606 i5NPI 5 R GACTGTTTCTCATCCTGTTATTATTTGT 224 rs1736442 i5NPI 5 R AACACACAGAAACATCAAGCTGAGC 225 rs1821380 i5NPI 5 R TTCCTGACATTCTCCTTCTTCTATCTG 226 rs1886510 i5NPI 5 R TATGACGCCTGGATTTTCACAACAAC 227 rs1979255 i5NPI 5 R CAGAGACTATGGATGGTATTTAGGTCAA 228 rs2016276 i5NPI 5 R ACTTTGTGTGGCTGAGAGAGAGAAA 229 rs2040411 i5NPI 5 R TGAGTGTTCTCTGTATTTTCTTACTCTAAG 230 rs2046361 i5NPI 5 R2 ATTTTTGGTCATTGTTGACACTTCACC 231 rs2056277 i5NPI 5 R2 GGTGTTAGGGAGACAGGCATGAATG 232 rs2076848 i5NPI 5 R TGAAACTTTTCAACTCTCCTACCGCC 233 rs2107612 i5NPI 5 R GTTAAAATTGCCACTAATTATGTGTTTT 234 rs2111980 i5NPI 5 R AACTGATCCTATGCAGCAAGATCTTTG 235 rs214955 i5NPI 5 R GATGCTTGCAAACAAAGACTGAAAAGG 236 rs221956 i5NPI 5 R GTCTGTGTGTCCTCTGAGATGATGAATG 237 rs2342747 i5NPI 5 R GGGAGGAAGAAAACAGAGAGTCTTGA 238 rs2399332 i5NPI 5 R AGTTTGTTGGCTTCTTTTGAGAAGTATC 239 rs251934 i5NPI 5 R GGCAGATGAAGTAGTAGATATCTGGCTG 240 rs279844 i5NPI 5 R GTTCAGTGTCAATTTTGACCAGATATT 241 rs2830795 i5NPI 5 R AGACATAGGACACACCATTTTATTGTCT 242 rs2831700 i5NPI 5 R TCAAAATATTTGGCTAAACTATTGCCGG 243 rs2920816 i5NPI 5 R2 CTGGAGTTATTAATAAATTGGATTATATAGC 244 rs321198 i5NPI 5 R TTACCTGTTTTCCTTTTGTGATTCCAC 245 rs338882 i5NPI 5 R ACCAAGTCAAGAGCTCTGAGAGACAT 246 rs354439 i5NPI 5 R ACAGTGAATGATATTCAGAATATTGTGC 247 rs3780962 i5NPI 5 R GAACAAGGTCAAGATATCAGCTTTCACC 248 rs430046 i5NPI 5 R AGGTCATACAATGAATGGTGTGATGT 249 rs4364205 i5NPI 5 R ATCCACCCATGAGAAATATATCCACAA 250 rs445251 i5NPI 5 R ACAATTCAAATTAATGTAAAAACTGCAAGTG 251 rs4606077 i5NPI 5 R TAGTTCTAGTGTGGGATCTGACTCC 252 rs560681 i5NPI 5 R GAACATCTGTTCAGGTTTCTCTCCATC 253 rs6444724 i5NPI 5 R GAAAGGACTAAATTGTTGAACACTGGT 254 rs6811238 i5NPI 5 R TGTGTGTTTTAAAGCCAGGTTTGTT 255 rs7041158 i5NPI 5 R GATGGACTGGAACTGAGGATTTTCA 256 rs717302 i5NPI 5 R AGCTTTAGAAAGGCATATCGTATTAACTG 257 rs719366 i5NPI 5 R TTATAGTGAGTAAAGGACAGGCCCC 258 rs722098 i5NPI 5 R2 ACACATCTGTTGACAGTAATGAAATATCC 259 rs722290 i5NPI 5 R GTTTAAACTTGGATACCATCCCCAAGAC 260 rs727811 i5NPI 5 R ATGAGATTGCTGGGAGATGCAGATG 261 rs729172 i5NPI 5 R CACATTTCCCTCTTGCGGTTACATAC 262 rs733164 i5NPI 5 R GACAAGCCTCGCTTGAGTTTTCTTT 263 rs735155 i5NPI 5 R TGTGAGAGTGTCACCGAATTCAACG 264 rs740598 i5NPI 5 R AAATAGCAATGGCTCGTCTATGGTTAG 265 rs740910 i5NPI 5 R TGCTAAGTAAGGTGAGTGGTATAATCA 266 rs763869 i5NPI 5 R ATAAATATGATGTGGCTACTCCCTCAT 267 rs8037429 i5NPI 5 R GCTACACCTCCATAGTAATAATGTAAGAG 268 rs8078417 i5NPI 5 R TGAAGCAGCTAGAGAACTCTGTACGT 269 rs826472 i5NPI 5 R2 TTTTGTCTCTGTTATATTAGTCACCTATCTC 270 rs873196 i5NPI 5 R ATAGCCCTGCATTCAAATCCCAAGTG 271 rs876724 i5NPI 5 R TCCATTTTTATACCACTGCACTGAAG 272 rs891700 i5NPI 5 R2 GCAGTAAAACATTTTCATCAAATTTCCA 273 rs901398 i5NPI 5 R TCTGGGTGCAAACTAGCTGAATATCAG 274 rs907100 i5NPI 5 R GAAAATCTGGAGGCAATTCATGATGCC 275 rs914165 i5NPI 5 R ATACAATGATGATCACACGGGACCCT 276 rs917118 i5NPI 5 R CCATGAAGATGGAGTCAACATTTTACA 277 rs938283 i5NPI 5 R TCCTAACCCCTAGTACGTTAGATGTG 278 rs964681 i5NPI 5 R GAGGTGATTTCTGTGAGGAACGTCG 279 rs987640 i5NPI 5 R GTACATTCACTTAACAGGCTCTCTTTCC 280 rs9905977 i5NPI 5 R AATTCATGAGCTGGTGTCCAAGGAG 281 rs993934 i5NPI 5 R ATAACAGTCTCCAGAGTATATTAGCTTAG 282 rs9951171 i5NPI 5 R GTTCCTCTGGGATGCAACATGAGAG 283 rs10497191 a5NPI T F GAAAGGATGAAGAGGGTGGATATTGGAG 284 rs1079597 a5NPI T F CCAAACCTCATCATCTCTTACCTGGATT 285 rs11652805 a5NPI T F GTCCAAAGTCAAGTGCAAGTATAGTTGG 286 rs1229984 a5NPI T F ACAATCTTTTCTGAATCTGAACAGCTTC 287 rs12439433 a5NPI T F CAAAGGAAGGCATTTCCTAATGATCTTC 288 rs12498138 a5NPI T F CTTTGCTTTGCTTTTCTTCTTCAGGGAA 289 rs12913832 p5NPI NU T F CTGCTTCAAGTGTATATAAACTCACAGT 290 rs1426654 a5NPI T F CCTAGGAAAGCAGTAACTAATTCAGGAG 291 rs1462906 a5NPI T F GCAATTTGTTCACTTTTAGTTTCGTAGC 292 rs1572018 a5NPI T F GGCCTAATATGCATGTGTTCATGTCTCT 293 rs16891982 a5NPI T F CAGAGTTTCTCATCTACGAAAGAGGAGT 294 rs174570 a5NPI T F ATCCTAGACCTCCAGGTGGAATGATC 295 rs17642714 a5NPI T F CTTGGCTGTCTCAATATTTTGGAGTAAG 296 rs1800414 a5NPI T F GAGTAAATGAGCTGTGGTTTCTCTCTTA 297 rs1834619 a5NPI T F CTTTCCATGTGGACCCTTTAACATTCAG 298 rs1876482 a5NPI T F GCATAGTGAGCTGTTGATAGAGCTTTTG 299 rs1919550 a5NPI T F CTAGAACAAAATCATTGGCTCTCCTAGT 300 rs192655 a5NPI T F GTCTGGTGAGTACTGGCTGAATGTAAA 301 rs200354 a5NPI T F CCAGAGGATGCTGCTAAACATTCTACAA 302 rs2024566 a5NPI T F GCTCATGCCTGGAATTCACCTTTATTTT 303 rs2042762 a5NPI T F CTAACTAGACATTTGGGCCACCTTACTT 304 rs2166624 a5NPI T F GTCTATGGTGCCTATAGAATGTACAGGT 305 rs2196051 a5NPI T F CCCTCTCAAGTTTGTGAGCAAATATCAC 306 rs2238151 a5NPI T F CTCTATCTTGCTGCAATGGACTTTCC 307 rs260690 a5NPI T F CCTAGAAACAGATTTTGAAGGGCTCTTG 308 rs2814778 a5NPI T F AAATGAGGGGCATAGGGATAAGGGA 309 rs310644 a5NPI T F CCTAGAAATCTGATACGTTATCCTATGA 310 rs3737576 a5NPI T F AGGAGAGATATATTCAACATGAACCCAA 311 rs3811801 a5NPI T F GAACATCTCTGACCAGAAATTTCCAGTA 312 rs3823159 a5NPI T F GTGTAGTGAAATCCTTAGACTTAGGTAA 313 rs3916235 a5NPI T F AATACATGAAAAAGTAATACATGGGGCA 314 rs4471745 a5NPI T F ATTAAATGTTTACTTCTATCTACAAGGA 315 rs4833103 a5NPI T F CATTTTGTGAAATGCAAAGGGCAAATCT 316 rs4891825 a5NPI NU T F GCTGAGAGGCTTAATTCCATCAAGATGA 317 rs4918664 a5NPI NU T F CCCATCCTAAACTTAGTTTTATGGGCAG 318 rs6754311 a5NPI T F GTAACACATTCTCTTTGGGAAGCTAGC 319 rs6990312 a5NPI NU T F CTTAGCTTCAGTGAAAATGGTTCCTCTC 320 rs7226659 a5NPI NU T F CTTTCTTAGCTCCTCTCCATTTCTCTTC 321 rs7326934 a5NPI NU T F GTCTATGCAGTGCTTCACTGAGGATTAT 322 rs735480 a5NPI NU T F CTCTATCTGCTCAGAGCCTGCTTAAAAG 323 rs7554936 a5NPI NU T F GGAAAGGATACAGTGTTGAGCAAGATAG 324 rs7657799 a5NPI NU T F GCCAACTTGATTCTCTTTCAAATGCTTG 325 rs7722456 a5NPI T F AGATGGGGTTTACCATGTTTCCCAG 326 rs798443 a5NPI T F GTACAGTAGTTAGTTTCCAGACTGATGA 327 rs7997709 a5NPI T F GTAAATATCTAACTGTGTTTCCCTCAGT 328 rs870347 a5NPI T F GAACCAAAAGGAATTAAGAGACTAGGGG 329 rs917115 a5NPI T F CTGCTTTTACGGCTTCTTCCTTTCTTC 330 rs10497191 a5NPI 5 R CCCACATCCTTCCCATTTATAGGCAA 331 rs1079597 a5NPI 5 R TACATGATCCTAAGGGCAGCAGGAA 332 rs11652805 a5NPI 5 R GTTTGGTGCATCCTCTTTCTCTCTC 333 rs1229984 a5NPI 5 R GACTGTAGTCACCCCTTCTCCAACA 334 rs12439433 a5NPI 5 R AGAGTGAAATACATAGAAAAGAAACTTAAAG 335 rs12498138 a5NPI 5 R ATTTGCGAGAAACAGATAAATATTGAAG 336 rs12913832 p5NPI NU 5 R ACAGGAACAAAGAATTTGTTCTTCATGG 337 rs1426654 a5NPI 5 R CCTTGGATTGTCTCAGGATGTTGCA 338 rs1462906 a5NPI 5 R CTGGGATGTTTGTTTTGGCTTTGTG 339 rs1572018 a5NPI 5 R ATTGGTAGTACACTAATGGATATATGTGAG 340 rs16891982 a5NPI 5 R GAATAAAGTGAGGAAAACACGGAGTTG 341 rs174570 a5NPI 5 R GAGAGAGGCAGAAAGGAGGGATGAA 342 rs17642714 a5NPI 5 R TACTCTGTCTTCAGTAGCTGTTTCTTGG 343 rs1800414 a5NPI 5 R TTAGACTCACCAAGATCAAGATGAATGC 344 rs1834619 a5NPI 5 R ATCTCAATAAAGCTGTTCAAAACAGAAAG 345 rs1876482 a5NPI 5 R TAAAGAAAATGCCATGGGCTGTACCC 346 rs1919550 a5NPI 5 R ATTGTGCAGCAGAACAGAGTGTAGTG 347 rs192655 a5NPI 5 R ATTCTTTGCATAGCTCACGAAATTTCCC 348 rs200354 a5NPI 5 R AAAATGAGACCTCGTATCTTTGCAGC 349 rs2024566 a5NPI 5 R AAATGCAGAACTGCCAAAAGAAACCC 350 rs2042762 a5NPI 5 R GAGAATCTGTGAATGCCAGGGTCTG 351 rs2166624 a5NPI 5 R ATGGATTCATGTTTCAGACATCTAATT 352 rs2196051 a5NPI 5 R ATCACTAGAAAGAAAAGAGTTCCTATTC 353 rs2238151 a5NPI 5 R GAAGTTTAAAAGAGTGGGAACATGGGG 354 rs260690 a5NPI 5 R CTACGTAAGCAAAAATGATCACGCAC 355 rs2814778 a5NPI 5 R AACCTGATGGCCCTCATTAGTCCTT 356 rs310644 a5NPI 5 R CACCAGATTTCTAGGAATAGCATGTGAG 357 rs3737576 a5NPI 5 R AAGAGCATAGTGAGGGGTTAGACCT 358 rs3811801 a5NPI 5 R CTTTATATTTAGTGTAGAGATCAGTCTCC 359 rs3823159 a5NPI 5 R TGAGTCCTTTACCTAATCTTGGTTGTC 360 rs3916235 a5NPI 5 R AATCCAAAGCAACTCTCTTTTCACCAC 361 rs4471745 a5NPI 5 R TTTACTGGAACCCTGATTTTGTTGGA 362 rs4833103 a5NPI 5 R TGCCACTGATATATCAGTACCTGAGT 363 rs4891825 a5NPI NU 5 R ACAATCTCAATCCCCCTTAATGTTTTC 364 rs4918664 a5NPI NU 5 R GTGGGCAGAGAGAGTAAGAGAACCT 365 rs6754311 a5NPI 5 R CAAACCAGATTCTGGCAGAATAGTTAGC 366 rs6990312 a5NPI NU 5 R CTTCTCTCCCATCCTCCTTCTCCAC 367 rs7226659 a5NPI NU 5 R AGATCAAGGGATCTGTGGGACAATAAC 368 rs7326934 a5NPI NU 5 R GGGGAGTGATTTCAAGCATCCTGATT 369 rs735480 a5NPI NU 5 R CATGAGTTTGAGGTAAGATGAAGGAGA 370 rs7554936 a5NPI NU 5 R TCTCTCTCATCCTAGTGAATGCCATC 371 rs7657799 a5NPI NU 5 R GGGTGATGATCTACCTTGCAGGTATA 372 rs7722456 a5NPI 5 R CTCAAGGCCCTGGGTCTGAAATTAC 373 rs798443 a5NPI 5 R ACATCTCCAGTTAATAATTTCCACTAAC 374 rs7997709 a5NPI 5 R TGGATTGCTCAACAAATAGTGCTAAAA 375 rs870347 a5NPI 5 R CATGCGACATCCAGGTAGCTAAAATAC 376 rs917115 a5NPI 5 R ATGGATAAAAATGGAACTTTCAAGAGAA 377 rs12203592 p5NPI T F GTTTTATGTAAAGCTTCGTCATATGGCT 378 rs12821256 p5NPI T F GTTCCAACTTAGTCATAAAGTTCCCTGG 379 rs12896399 p5NPI T F GGGTCTTGATGTTGTATTGATGAGGAAG 380 rs1393350 p5NPI T F CCTAACAGAAAGTCACTGTTTGTATCTG 381 rs1800407 p5NPI T F TCACTCTGGCTTGTACTCTCTCTGTG 382 rs2378249 p5NPI T F GGCTGGTTTCAGTCTGGAGACTTTATTT 383 rs2402130 p5NPI T F CTTCACCTCGATGACGATGATGATGAT 384 rs4959270 p5NPI T F GACAATAACAGCACAAAGGATGGAAAAG 385 rs1805009 p5NPI T F GAACCAGACCACACAATATCACCAC 386 rs28777 p5NPI T F TCTACCTCTTTGATGTCCCCTTCGATAG 387 rs16891982 p5NPI T F CAGAGTTTCTCATCTACGAAAGAGGAGT 388 rs683 p5NPI T F CCCAGCTTTGAAAAGTATGCCTAGAACT 389 rs12913832 p5NPI T F CTGCTTCAAGTGTATATAAACTCACAGT 390 rs12203592 p5NPI 5 R TTGTTTCATCCACTTTGGTGGGTAAAAG 391 rs12821256 p5NPI 5 R TAATTAAGCTCTGTGTTTAGGGTTTTT 392 rs12896399 p5NPI 5 R CAATTCTTTGTTCTTTAGGTCAGTATAT 393 rs1393350 p5NPI 5 R TACTCTTCCTCAGTCCCTTCTCTGC 394 rs1800407 p5NPI 5 R TGAGACAGAGCATGATGATCATGGC 395 rs2378249 p5NPI 5 R GCACAAGTCTAGGAACTACTTTGCAC 396 rs2402130 p5NPI 5 R GAAGTATTTGAACCATACGGAGCCC 397 rs4959270 p5NPI 5 R TGAGGAACACATCCAAACTATGACAC 398 rs1805009 p5NPI 5 R TTTCTCGCCCTCATCATCTGCAATG 399 rs28777 p5NPI 5 R TCAGTTGATTTCATGTGATCCTCACAG 400 rs16891982 p5NPI 5 R GAATAAAGTGAGGAAAACACGGAGTTG 401 rs683 p5NPI 5 R ATTACCTTCTTTCTAATACAAGCATATG 402 rs12913832 p5NPI 5 R ACAGGAACAAAGAATTTGTTCTTCATGG

Example 2 DNA Profiling for Databanking

This example describes an experiment following the workflow of FIG. 2 . This example does not utilize UMIs, as it could be assumed that the samples obtained are from individuals whose identity is already known.

For this experiment, STRs are multiplexed with iSNPs as found in Table 3.

TABLE 3 Identity informative SNPs and STRs Identity informative 5NPs rs1005533 rs1357617 rs2076848 rs4530059 rs763869 rs10092491 rs1360288 rs2107612 rs4606077 rs8037429 rs1015250 rs1382387 rs2111980 rs560681 rs8078417 rs1024116 rs1413212 rs214955 rs576261 rs826472 rs1028528 rs1454361 rs221956 rs6444724 rs873196 rs1029047 rs1463729 rs2269355 rs6811238 rs876724 rs1031825 rs1490413 rs2342747 rs6955448 rs891700 rs10488710 rs1493232 rs2399332 rs7041158 rs901398 rs10495407 rs1498553 rs251934 rs717302 rs907100 rs1058083 rs1523537 rs279844 rs719366 rs914165 rs10773760 rs1528460 rs2830795 rs722098 rs917118 rs10776839 rs159606 rs2831700 rs722290 rs938283 rs1109037 rs1736442 rs2920816 rs727811 rs964681 rs1294331 rs1821380 rs321198 rs729172 rs987640 rs12997453 rs1886510 rs338882 rs733164 rs9905977 rs13182883 rs1979255 rs354439 rs735155 rs993934 rs13218440 rs2016276 rs3780962 rs737681 rs9951171 rs1335873 rs2040411 rs430046 rs740598 rs1336071 rs2046361 rs4364205 rs740910 rs1355366 rs2056277 rs445251 rs7520386 Autosomal 5TRs 0151656 C5F1PO vWA 021511 0452408 025441 075820 0135317 TPOX 01751301 0251338 0851179 Penta E 5E33 0951122 0351358 01051248 0165539 Penta 0 0651043 FGA TH01 018551 02251045 Amelogenin 055818 0125391 0195433 0205482 X 5TRs OX58378 OX58377 OX510101 OX510148 OX510146 OX57132 OX510135 OX510134 OX510079 HPRTB OX510074 OX57423 OX510103 Y 5TRs OY5456 OY5393 OY5437 OY5533 OY5449 OY53891/II OY5391 OY5438 OY5518 OY5522 OY5390 OY5439 OY5448 OY5570 OY5505 OY5458 OY5635 OY5576 OY5643 OY5627 OY519 OY5392 OY5481 OY5460 OYF38751a/b OY5385a/b YGATAH4 OY5549 OY5612

Additional SNPs and STRs could of course be added to the above list. Examples of other potential targets include, but are not limited to, those markers found in Table 4.

TABLE 4 Examples of additional STRs and SNPs for multiplexing Identity informative 5NPs rs1004357 rs1554472 rs2567608 rs521861 rs9606186 rs1019029 rs1872575 rs2811231 rs5746846 rs985492 rs1027895 rs2073383 rs2833736 rs590162 rs9866013 rs10500617 rs2175957 rs315791 rs6591147 rs10768550 rs2255301 rs3744163 rs689512 rs12480506 rs2270529 rs4288409 rs7205345 rs13134862 rs2272998 rs464663 rs7229946 rs1358856 rs2291395 rs4789798 rs7704770 rs1410059 rs2292972 rs4796362 rs8070085 rs1478829 rs2503107 rs4847034 rs9546538 Autosomal 5TRs 0151677 0354529 0185853 01051435 01154463 0651017 01451434 0552500 0151627 01GATA113 0251776

Primers were designed to contain a gene-specific PCR primer sequence at the 3′ end and an adapter tag sequence at the 5′ end. In this experiment, the forward primers contain the tag sequence for the TruSeq Custom Amplicon i5 adapters and the reverse primers contain the tag sequence for the TruSeq Small RNA kit i7 adapters. The tags can be used as amplification primer sites as well as sequencing primer sites.

Primers were designed to contain a gene-specific PCR primer sequence at the 3′ end and an adapter tag sequence at the 5′ end. In this experiment, the forward primers contain the tag sequence for the TruSeq® Custom Amplicon kit (an amplicon sequencing kit) i5 adapters and the reverse primers contain the tag sequence for the TruSeq® Small RNA kit (an RNA library preparation kit) i7 adapters. The tags can be used as amplification primer sites as well as sequencing primer sites.

Adapter i5 tag sequence (SEQ ID NO: 403) 5′TACACGACGCTCTTCCGATCT3′ Adapter i7 tag sequence (SEQ ID NO: 404) 5′CTTGGCACCCGAGAATTCCA3′

To balance the amplification between the STRs and the SNPs in the multiplex, primer design parameters were modified for the SNPs as described in Example 1. The original set of SNP primers designed using Illumina's Design Studio were classic PCR primers-short sequences with high melting temperatures and little to no secondary structure. Design Studio was used to design TruSeq® Custom Amplicon kit (an amplicon sequencing kit) Probes and to create the reverse complement of the down-stream probe to make the reverse PCR primer. These primers, however, did not multiplex well and one bad primer could turn the assay from good to bad (e.g., all primer-dimer and no product) (FIG. 4 .) In an attempt to create better primers for multiplexing, Primer3 (shareware) was used that contains a mispriming library feature. It was discovered that the Primer3 designed primers performed even more poorly in the multiplex assay than the Design Studio primers. Surprisingly, data were being generated showing that the STR primers were multiplexing well. It was observed that the poorly designed primer pairs directed to STR targets did not cause multiplex failures as the SNP primers did. The STR primers are long, AT-rich, and have low melting temperatures, contrary to what is known as a “good” primer.

The SNP primers were redesigned following the parameters of Example 1. The primers were mixed together for all of the targets. For this example, primer pairs for 56 STRs were mixed with primer pairs for 75 iSNPs, aSNPs, and phenotypic-informative SNPs. Polymerase (PHUSION HOT START II in this example) was added to a mastermix of all of the components required for PCR and the primers were added. The mix was pipetted into wells of a PCR plate, but the amplification could also be performed in tubes, etc. DNA was added to the plate as purified DNA in 15 microliter volume, however lysed extracts of blood or buccal samples from swabs or non-treated filter paper, or directly from blood or buccal samples on FT A Cards, etc. could also be used. For this experiment, purified control2800M DNA at 1 ng and 100 pg was used. The reactions were subjected to PCR for a determined number of cycles (in the case of the example, 25cycles) following the protocol:

95° C. 96° C. 54° C. 68° C. 3 min 1 min 2 min* 45 sec** 60° C.  4° C. 30 min hold *ramp 0.5° C./sex **ramp 0.2° C./sec-

After cycling, the plates were removed from the thermal cycler. The reaction was brought to 50 microliters with polymerase (Kapa HiFi, Kapa Biosystems), PCR mastermix containing all of the components required for PCR, and a pair of adapters (one i7 and one i5 adapter). A second round of PCR was performed for a determined number of cycles (10 cycles in the case of the example) to generate the sequencing libraries, following the protocol:

98° C. 30 sec 98° C. 15 sec 66° C. 30 sec 72° C.  1 min 72° C.  5 min 10° C. hold

After cycling, the plate containing the completed libraries was removed from the thermal cycler. At this point, the samples can be pooled by volume and purified as a single sample using magnetic beads (SPRI) for example. The samples also can be purified individually. The pool or the individual libraries can be quantified by using a qPCR-based method, by using a Fragment Analyzer or a microfluidics-based automated electrophoresis method, e.g., BIOANALYZER, or by using a quantitation kit, e.g., PicoGreen™ and a plate reader (as in the case of the example). A skilled artisan will know the myriad of options for library quantitation. If the libraries are purified individually, they can be normalized to 2 nM each concentration and pooled by volume.

The pools of purified libraries were denatured, diluted, clustered and sequenced on the MISEQ System (a sequencing instrument) with a 350-cycle sequencing run and the two index reads. After sequencing, the samples were demultiplexed according to the adapter sequences and analyzed through the Forensics Genomics pipeline (Illumina, Inc.). The STR reads were separated from the SNP reads and analyzed independently. The STRs were analyzed using the algorithm described in a previous patent application (PCT/US2013/30867, incorporated herein by reference in its entirety). The repeat number(s) and any sequence variations were reported along with the read numbers. The SNPs were analyzed using a manifest and the calls were reported along with the read numbers. The relative balance between alleles (Min/Max %), balance between loci (% CV), error rates, and stutter rates were calculated for the STR loci. The results for the STRs in the initial databanking multiplex are shown in FIG. 5A-C. The balance (mean balance 80%), stutter (3%) and error rates (less than 5%) meet design input requirements for the loci included in this example. The % CV (142%) was calculated using all 56 loci. Even though the primers used show inter-locus balance, further primer optimization is anticipated to improve the inter-locus balance. The calls for the known loci match published results for 2800M. The results for the SNPs are shown in FIG. 5D-E. The coverage, allele calls, stutter and other artifacts for the 56 STR loci in the large multiplex are shown in FIG. 6 . These graphs mimic the electropherograms generated by CE technology. The bars are analogous to peaks for the defined allele (X axis) and the read counts (Y axis) are analogous to RFU. The coverage for the SNPs was anywhere from 10-2500X depending on the SNP, however every SNP that was multiplexed was counted and provided accurate calls.

The pools of purified libraries were denatured, diluted, clustered and sequenced on the MiSeq sequencing instrument with a 350-cycle sequencing run and the two index reads. After sequencing, the samples were demultiplexed according to the adapter sequences and analyzed through the Forensics Genomics pipeline (Illumina, Inc.). The STR reads were separated from the SNP reads and analyzed independently. The STRs were analyzed using the algorithm described in a previous patent application (PCT/US2013/30867, incorporated herein by reference in its entirety). The repeat number(s) and any sequence variations were reported along with the read numbers. The SNPs were analyzed using a manifest and the calls were reported along with the read numbers. The relative balance between alleles (Min/Max %), balance between loci (% CV), error rates, and stutter rates were calculated for the STR loci. The results for the STRs in the initial databanking multiplex are shown in FIG. 5A-C. The balance (mean balance 80%), stutter (3%) and error rates (less than 5%) meet design input requirements for the loci included in this example. The % CV (142%) was calculated using all 56 loci. Even though the primers used show inter-locus balance, further primer optimization is anticipated to improve the inter-locus balance. The calls for the known loci match published results for 2800M. The results for the SNPs are shown in FIG. 5D-E. The coverage, allele calls, stutter and other artifacts for the 56 STR loci in the large multiplex are shown in FIG. 6 . These graphs mimic the electropherograms generated by CE technology. The bars are analogous to peaks for the defined allele (X axis) and the read counts (Y axis) are analogous to RFU. The coverage for the SNPs was anywhere from 10-2500× depending on the SNP, however every SNP that was multiplexed was counted and provided accurate calls.

Example 3 DNA Profiling for Criminal Casework

This example describes an experiment following the workflow of FIG. 3 . This example incorporates UMIs into the primers, as it could be assumed that the samples obtained are from individuals whose identity is not already known.

For this experiment, the STRs were multiplexed with iSNPs, aSNPs and phenotypic-informative SNPs as found in Table 5.

TABLE 5 case work STRs and SNPs Identity informative SNPs rs1005533 rs1357617 rs2076848 rs4530059 rs763869 rs10092491 rs1360288 rs2107612 rs4606077 rs8037429 rs1015250 rs1382387 rs2111980 rs560681 rs8078417 rs1024116 rs1413212 rs214955 rs576261 rs826472 rs1028528 rs1454361 rs221956 rs6444724 rs873196 rs1029047 rs1463729 rs2269355 rs6811238 rs876724 rs1031825 rs1490413 rs2342747 rs6955448 rs891700 rs10488710 rs1493232 rs2399332 rs7041158 rs901398 rs10495407 rs1498553 rs251934 rs717302 rs907100 rs1058083 rs1523537 rs279844 rs719366 rs914165 rs10773760 rs1528460 rs2830795 rs722098 rs917118 rs10776839 rs159606 rs2831700 rs722290 rs938283 rs1109037 rs1736442 rs2920816 rs727811 rs964681 rs1294331 rs1821380 rs321198 rs729172 rs987640 rs12997453 rs1886510 rs338882 rs733164 rs9905977 rs13182883 rs1979255 rs354439 rs735155 rs993934 rs13218440 rs2016276 rs3780962 rs737681 rs9951171 rs1335873 rs2040411 rs430046 rs740598 rs1336071 rs2046361 rs4364205 rs740910 rs1355366 rs2056277 rs445251 rs7520386 Autosomal 5TRs 0151656 C5F1PO vWA 021511 0452408 025441 075820 0135317 TPOX 01751301 0251338 0851179 Penta E 5E33 0951122 0351358 01051248 0165539 Penta 0 0651043 FGA TH01 018551 02251045 Amelogenin 055818 0125391 0195433 0205482 X 5TRs OX58378 OX58377 OX510101 OX510148 OX510146 OX57132 OX510135 OX510134 OX510079 HPRTB OX510074 OX57423 OX510103 Y5TRs OY5456 OY5393 OY5437 OY5533 OY5449 OY53891/II OY5391 OY5438 OY5518 OY5522 OY5390 OY5439 OY5448 OY5570 OY5505 OY5458 OY5635 OY5576 OY5643 OY5627 OY519 OY5392 OY5481 OY5460 OYF38751a/b OY5385a/b YGATAH4 OY5549 OY5612 Phenotypic informative 5NPs N29insA rs1805006 rs1110400 rs12203592 rs2378249 rs11547464 rs1805007 rs28777 rs1042602 rs12896399 rs885479 rs1805009 rs16891982 rs1800407 rs1393350 rs1805008 Y1520CH rs12821256 rs2402130 rs683 rs1805005 rs2228479 rs4959270 rs12913832 Ancestry informative 5NPs rs10497191 rs17642714 rs2238151 rs4471745 rs7554936 rs1079597 rs1800414 rs2593595 rs459920 rs7657799 rs11652805 rs1834619 rs260690 rs4833103 rs7722456 rs1229984 rs1871534 rs2814778 rs4891825 rs798443 rs12439433 rs1876482 rs310644 rs4918664 rs7997709 rs12498138 rs1919550 rs3737576 rs671 rs870347 rs12913832 rs192655 rs3811801 rs6754311 rs917115 rs1426654 rs200354 rs3814134 rs6990312 rs9522149 rs1462906 rs2024566 rs3823159 rs7226659 rs1572018 rs2042762 rs3827760 rs7251928 rs16891982 rs2166624 rs3916235 rs7326934 rs174570 rs2196051 rs4411548 rs735480

This example includes UMIs for the STR primers. For these examples, only the STR primers contain UMIs, however both STR and SNP primers could include UMIs if desired and that option is not excluded from practice. For this example however only the STR primers incorporate UMIs for demonstration purposes. Unique molecule identifiers were introduced during two cycles of PCR (FIG. 3 ). First, as for Example 2, the PCR primers contain a gene-specific PCR primer sequence at the 3′ end and an adapter tag sequence at the 5′ end, the same as for tag sequences used in Example 2 for i5 and i7 sequences. In this experiment, the UMIs are positioned between the gene-specific primer sequence and the tag sequence. In the case of this example, there were five randomized bases used for the UMI on both the forward and reverse primers. The primers were mixed together for all of the targets. The primer mix comprised of 26 Autosomal STR primer pairs and 86 SNP primer pairs (92 SNPs covered). Polymerase (Hot-start Phusion II in this example) was added to a mastermix of all of the components required for PCR, and the primers were added. The mix was pipetted into wells of a PCR plate. DNA was added to the plate as purified DNA, optimally 1 ng. As in Example 2, purified DNA from 2800M control was tested at 1 ng. The multiplex reaction mixture was subjected to two cycles of PCR following the protocol:

98 C.  3 min 98 C.  2 min 54 C. 12 min ramp0.2 C./sec 72 C.  4 min 2 cycles  4 C. hold

After cycling, the samples were removed from the thermal cycler and E. coli single-stranded DNA binding protein (SSB) was added to the reaction. It was contemplated that the SSB reduces primer dimers by the unused tagged gene-specific primers and prevents any more amplification from these primers. The SSB was incubated with the sample on ice, alternatively RT or 37C incubation could also be used. After this incubation, polymerase (PHUSION HOT START II in this example) was added to a mastermix of all of the components required for PCR, and the mastermix was added to the sample with a pair of adapters (i7 and i5 adapters) and cycled for a determined number of cycles (in this experiment 34 cycles), following the protocol:

95 C. 3 min 95 C. 30 sec 66 C. 30 sec 72 C. 1 min 72 C. 5 min I0 C. hold

The samples were purified with SPRI beads, and the individual libraries could be quantified by using a qPCR-based method, by using a Fragment Analyzer (as in the case of the example) or a microfluidics-based automated electrophoresis method, e.g., BIOANALYZER, or by using a quantitation kit, e.g., PicoGreen™ and a plate reader. The libraries were normalized to 2 nM each concentration and pooled by volume.

The pools of purified libraries were denatured, diluted, clustered and sequenced using the MISEQ System (a sequencing instrument) with a 350×100-cycle sequencing run and the two index reads. After sequencing, data was determined as reported in Example 2. However, since the primers contain UMIs, the UMIs were used to collapse the data by using PCR duplicates to remove sequencing and PCR errors and artifacts. The SNPs were analyzed using a manifest and the calls were reported along with the read numbers. The relative balance between alleles (Min/Max %), balance between loci (% CV), error rates, and stutter rates (STRs, only) were calculated. The results for the initial casework multiplex are shown in FIG. 7A-E. The coverage, allele calls, stutter and other artifacts for the 26 STR loci in the large multiplex are shown in FIG. 8 . These graphs mimic the electropherograms generated by CE. The bars are analogous to peaks and the read counts are analogous to RFU. The coverage for the SNPs was anywhere from 10-5500X depending on the SNP, however every SNP that was multiplexed was counted and provided useful results.

One result that was generated by these studies was that stutter was shown to be a PCR artifact. This has been hypothesized by many investigators (and polymerase slippage has been indicated in human colon cancers), but this hasn't been demonstrated for the Forensics assays. The UMIs can be used to show that stutter is indeed a PCR artifact. The products with n+1 or n-1 repeats have the same UMIs as the products with the correct number of repeats (FIG. 9 ). In FIG. 9A each locus shows the results without UMI correction compared to FIG. 9B where UMI correction is performed. As demonstrated, without UMI correction the balance between the alleles is not as good as when UMI correction is performed. Further, there is considerably more stutter that is apparent without UMI correction. The portion of the bar above the inter-bar line represents the sequencing error whereas below the line represents the correct sequence within the STR sequence. The error is greatly reduced with UMI correction. For example, SE33 locus has error that is removed with UMI correction. Error correction can be extremely important for criminal casework to provide the most accurate DNA profiling possible.

Example 4 DNA Profiling Using 12 Sample Individuals

Methods and Material

DNA from 12 sample individuals (Sample#: 1, 3, 4, 5, 6, 7, 10, 13, 14, 15, 16, 17) and one reference genome (2800M) was tested following the workflow of FIG. 3 . This experiment incorporates UMIs into the STR primers as described in Example 3. Two replicates of each sample were analyzed with the Illumina® ForenSeq DNA Signature Library Prep Kit on the MISEQ System (a sequencing instrument) sequencer. One ng DNA was used for each replication using the DNA Primer Mix B: Collected samples mix, which contains primers for 61 STRs plus Amelogenin, 95 identity-informative SNPs, 56 ancestry-informative SNPs, 22 phenotypic-informative SNPs (2 ancestry SNPs are also used for phenotype prediction).

Default Settings

STR: analytical threshold=6.5%; interpretation threshold=15%. SNP: analytical threshold=3%; interpretation threshold=15%.

The high level sequencing calls, such as coverage and loci called, for the DNA profiling of the 12 sample individuals are shown in FIG. 12 . As can be seen, every locus was covered by at least 100,000 reads in both replications. Only two samples resulted in a failed STR call (1 out of 61). All of the 173 SNPs were successfully called in all individuals in both replications. Sample STR calls of two sample individuals are shown in FIG. 16 . Sample SNP calls of two sample individuals are shown in FIG. 17 . FIG. 13 shows the population statistics, such as the random match probability (RMP) of the National Institute of Standards and Technology (NIST) auto-STRs, 95% confidence haplotype frequency of NIST Y-STRs, RMP of the dbSNP iSNPs, and the RMP of the STRs from the U.S. Y-STR database.

Phenotypes, such as eye color and hair color of the 12 sample individuals and the reference individual were predicted based on the genotype of pSNPs in the experiment, and compared to the self-reported phenotypes (FIG. 14 ). High degree of correlation between the predicted and reported phenotypes was observed.

Ancestry of the 12 sample individuals were predicted using the genotype of 56 aSNPs in the experiment. PCA1 and PCA3 scores of each sample individual were calculated, and plotted against reference samples on an ancestry plot. As shown in FIG. 15 , ancestry of the sample individuals can be predicted based on the location on the ancestry plot. Fourteen centroid points were included in the ancestry plot (circles). Based on the closest centroid point, the ancestry of each sample individual was predicted.

The DNA profiling experiment also showed high level of intra-locus balance in both the STR loci and the SNP loci, as can be seen in FIG. 18 , and low level of stutter, as can be seen in FIG. 19 .

Six of the 12 individuals plus 2800M have at least one isometric heterozygote locus, which is shown in FIG. 20 . An isometric heterozygote locus is defined as an STR that has the same repeat number, two different sequences, which are equally balanced. Using the information on the variants in the STR D8 S1179, the 13 allele of Sample 15 was traced to the grandmother, Sample 17 (FIG. 21 ). Similar variant information in the STR D 13S317 was used to trace the alleles of Sample 15. However, in this case the origin of either allele cannot be ascertained (FIG. 22 ).

Example 5 DNA Profiling for Research, Forensic, or Paternity Use

This example is based on the workflow described in ForenSeg™ DNA Signature Prep Guide (Illumina, San Diego, Calif.), the content of which is hereby incorporated by reference in its entirety.

Either purified DNA or crude lysate may be used for this example. For purified DNA, each 1 ng sample is diluted to 0.2 ng/μl with nuclease-free water. For crude lysate, each 2 μl sample is diluted with 3 μl nuclease-free water. A Master Mix is set up for eight or more reactions. For each reaction, 5.4 μl of ForenSeq™ PORI Reaction Mix, 0.4 μl of ForenSeq™ Enzyme Mix and 5.8 μl of DNA primer Mix (A or B) are added into a 1.5 ml microcentrifuge tube. 10 μl of Master Mix is transferred to each well of the PCR plate, and the DNA or lysate is added. The multiplex reaction mixture is subjected PCR following the protocol:

98 degrees C. for 3 min.

8 cycles of:

-   -   96 degrees C. for 45 sec     -   80 degrees C. for 30 sec     -   54 degrees C. for 2 min, with specified ramping mode     -   68 degrees C. for 2 min, with specified ramping mode         10 cycles of:     -   96 degrees C. for 30 sec     -   68 degrees C. for 3 min, with specified ramping mode         68 degrees C. for 10 min         Hold at 10 degrees C.

After cycling, the samples are removed from the thermal cycler. ForenSeq™ PCR2 Reaction Mix is added to the samples with a pair of adapters (i7 and i5 adapters) and cycled fora 15 cycles, following the protocol:

98 degrees C. for 30 sec.

15 cycles of:

-   -   98 degrees C. for 20 sec.     -   66 degrees C. for 30 sec.     -   68 degrees C. for 90 sec.         68 degrees C. for 10 min         Hold at 10 degrees C.

The samples are purified with Sample Purification Beads, and the libraries are normalized and pooled by volume. The pooled libraries are diluted in Hybridization Buffer (HT 1), added with Human Sequencing Control (HSC), and heat denatured in preparation for sequencing.

Example 6 Genotyping with Degraded DNA

FIG. 23 shows genotyping results using sheared and/or DNase-treated DNA representing degraded DNA As shown, more than 50% STR and SNP loci were correctly called with sheared DNA A random match probability (RMP) of 10⁻¹⁹ was achieved with DNA of less than 100 bp. Correct ancestry was also predicted using degraded DNA.

Example 7 Genotyping Sensitivity

FIGS. 24 and 25 show genotyping sensitivity results at sub-nanogram DNA input levels from 7.82 pg to 1 ng. As shown, 100% alleles were successfully called at 125 ng input DNA for both STR and SNP. More than 50% alleles were successfully called at as low as 7.82 pg input DNA Intra-locus balance was greater than 70% for most loci at 1 ng input DNA.

All numbers expressing quantities of ingredients, reaction conditions, and the like used in the specification are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth therein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Citation of the above publications or documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

Although the present invention has been fully described in connection with embodiments thereof with reference to the accompanying drawings, it is to be noted that various changes and modifications will become apparent to those skilled in the art. Such changes and modifications are to be understood as being included within the scope of the present invention. The various embodiments of the invention should be understood that they have been presented by way of example only, and not by way of limitation. Likewise, the various diagrams may depict an example architectural or other configuration for the invention, which is done to aid in understanding the features and functionality that can be included in the invention. The invention is not restricted to the illustrated example architectures or configurations, but can be implemented using a variety of alternative architectures and configurations. Additionally, although the invention is described above in terms of various exemplary embodiments and implementations, it should be understood that the various features and functionality described in one or more of the individual embodiments are not limited in their applicability to the particular embodiment with which they are described. They instead can, be applied, alone or in some combination, to one or more of the other embodiments of the invention, whether or not such embodiments are described, and whether or not such features are presented as being a part of a described embodiment. Thus the breadth and scope of the invention should not be limited by any of the above-described exemplary embodiments.

Terms and phrases used in this document, and embodiments thereof, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing: the term “including” should be read as meaning “including, without limitation” or the like; the term “example” is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; and adjectives such as “conventional,” “traditional,” “normal,” “standard,” “known”, and terms of similar meaning, should not be construed as limiting the item described to a given time period, or to an item available as of a given time. But instead these terms should be read to encompass conventional, traditional, normal, or standard technologies that may be available, known now, or at any time in the future. Likewise, a group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as “and/or” unless apparent from the context or expressly stated otherwise. Similarly, a group of items linked with the conjunction “or” should not be read as requiring mutual exclusivity among that group, but rather should also be read as “and/or” unless it is apparent from the context or expressly stated otherwise. Furthermore, although items, elements or components of the invention may be described or claimed in the singular, the plural is contemplated to be within the scope thereof unless limitation to the singular is explicitly stated. For example, “at least one” may refer to a single or plural and is not limited to either. The presence of broadening words and phrases such as “one or more,” “at least,” “but not limited to”, or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent. 

What is claimed is:
 1. A method for constructing a DNA profile comprising: providing a nucleic acid sample, amplifying the nucleic acid sample with a plurality of primers, wherein the primers in the plurality of primers specifically hybridize to (i) at least 30 target sequences each comprising a SNP and at least one target sequence comprising a tandem repeat sequence, or (ii) at least 24 target sequences each comprising a tandem repeat and at least one target sequence comprising a SNP, in a multiplex reaction to generate amplification products, and wherein at least one primer in the plurality of primers has a low melting temperature and also has a length of at least 24 nucleotides, and determining the genotypes of the SNP(s) and the tandem repeat(s) in the amplification products, thereby constructing the DNA profile.
 2. The method of claim 1, wherein the nucleic acid sample is from a human.
 3. The method of claim 1, wherein at least one SNP comprises a SNP that is known to indicate the ancestry or a phenotypic characteristic of the source of the nucleic acid sample.
 4. The method of claim 1, wherein at least one of the plurality of primers has a length of 24 to 38 nucleotides.
 5. The method of claim 4, wherein at least one of the plurality of primers has a melting temperature that is less than 60 degrees C.
 6. The method of claim 1, wherein amplifying the nucleic acid sample comprises performing polymerase chain reaction (PCR) on the nucleic acid sample.
 7. The method of claim 1, wherein the nucleic acid sample comprises genomic DNA.
 8. The method of claim 7, wherein providing a nucleic acid sample comprises providing a forensic sample comprising the nucleic acid.
 9. The method of claim 1, wherein determining the genotypes comprises determining at least 90% of the genotypes of the at least one SNP and at least one tandem repeat.
 10. The method of claim 1, wherein at least one of the plurality of primers comprises one or more tag sequences comprising a unique molecular identifier tag.
 11. A method of constructing a nucleic acid library, comprising: providing a nucleic acid sample, and amplifying the nucleic acid sample with a plurality of primers, wherein the primers in the plurality of primers specifically hybridize to at least one single nucleotide polymorphism (SNP) and at least one target sequence comprising a tandem repeat sequence in a multiplex reaction to generate amplification products, wherein at least one of the plurality of primers has a low melting temperature and also has a length of at least 24 nucleotides, and wherein at least one of the plurality of primers comprises one or more tag sequences comprising a primer tag, a capture tag, a sequencing tag, a unique molecular identifier tag, or a combination thereof; and amplifying the amplification products with a second plurality of primers.
 12. The method of claim 11, wherein the at least one SNP indicates the ancestry or a phenotypic characteristic of the source of the nucleic acid sample.
 13. The method of claim 11, wherein at least one primer in the second plurality of primers comprises a portion corresponding to the primer tag of the plurality of primers and one or more tag sequences.
 14. The method of claim 11, further comprising adding single-stranded binding protein (SSB) to the amplification products.
 15. The method of claim 11, wherein amplifying the nucleic acid sample comprises polymerase chain reaction (PCR) amplification.
 16. The method of claim 11, wherein the primers in the plurality of primers specifically hybridize to at least 30 target sequences each comprising a SNP.
 17. The method of claim 11, wherein the primers in the plurality of primers specifically hybridize to at least 24 target sequences each comprising a tandem repeat. 